VERTEBRATE  HISTOLOGY 


GAGE  AND  KINGSBURY 


UNIVERSITY  OF  CALIFORNIA 

MEDICAL  CENTER  LIBRARY 

SAN  FRANCISCO 


Gift  of  the 
Department  of  Anatomy,  U.C 


\ 


VERTEBRATE  HISTOLOGY 


A  GUIDE  FOR  COURSE  2,  DEPART- 
MENT OF  MICROSCOPY,  HISTOLOGY 
AND  EMBRYOLOGY.  CORNELL  UNI- 
VERSITY AND  THE  NEW  YORK  STATE 
VETERINARY  COLLEGE 


BY      SIMON      HENRY     GAGE 

PROFESSOR     OF    MICROSCOPY,      HISTOLOGY 
AND     EMBRYOLOGY 

AND 

BENJAMIN  FREEMAN  KINGSBURY 

AvSSISTANT     PROFESSOR      OF      MICROSCOPY 
HISTOLOGY    AND   EMBRYOLOGY 


COMSTOCK    PUBLISHING   COMPANY 

ITHACA,    NEW    YORK 
1899-1900 


Copyright,  1899. 
BY  GAGE  AND  KINGSBURY. 


CONTENTS. 


PAGE. 

Literature,  Equipment  and  Reagents 7-10 

I.    INTRODUCTORY u 

II.    WEEKLY  EXERCISES 13 

Introductory  and  Technic 15 

The  Epithelial  Tissues 18 

The  Connective  and  Supporting  Tissues 22 

The  Blood  and  Lymph 30 

The  Muscular  Tissues  and  the  Muscular  System 37 

The  Nervous  Tissues  and  The  Peripheral  Nervous  System 41 

The  Blood  and  Lymph  Vascular  System 45 

The  Digestive  System 49 

The  Respiratory  System 57 

The  Urinary  Organs 60 

The  Genital  Organs 63 

The  Skin  and  its  Appendages 67 

The  Central  Nervous  System 74 

The  Organs  of  Special  Sense 78 

III.     TECHNIC. 

Isolation 82 

Fixation 87 

Sectioning,  Free-hand  and  by 94 

(a)  The  Paraffin  Method 96 

(b)  The  Collodion  Method 99 

(c)  The  Freezing  Method 105 

Staining  and  Mounting Io6 

Staining IIJ 

Mounting J2o 

Sealing,  Labeling,  Cleaning  Slides  and  Covers 123 

Special  Methods I27 

Blood "7 

Fine  Injection I29 

Bone  and  Tooth,  (a)  and  (b) 130 

Nervous  System J32 

Silver  Nitrate  Impregnations 12& 

Formulas  (additional) J37 


PREFATORY  NOTE. 


This  guide  is  the  outgrowth  of  the  printed  and  mimeographed  sheets  fur- 
nished the  students  during  the  last  few  years  in  the  course  of  Vertebrate  His- 
tology in  Cornell  University.  The  course  is  meant  to  be  fairly  comprehensive 
in  order  to  meet  the  needs  of  the  three  groups  of  students  who  pursue  it, 
viz.  :  students  in  the  academic  department,  both  graduate  and  undergraduate, 
whose  aim  is  general  culture  and  attainment  of  a  sound  basis  for  investigation 
in  morphology  and  physiology;  (2)  Veterinary  students;  (3)  Medical  stu- 
dents. For  the  two  latter,  the  course  forms  an  integral  part  of  their  profes- 
sional training,  and  is  designed  to  furnish  aid  in  the  comprehension  of  gross 
anatomy,  of  physiology,  and  also  as  a  foundation  for  the  appreciation  of  the 
changes  revealed  by  pathological  histology. 

It  will  be  seen  in  looking  through  the  guide  that  definite  information  is 
given  or  will  be  supplied  during  the  course  for  each  specimen  studied,  and  that 
each  student  has  opportunity  and  is  required  to  carry  on  from  the  beginning 
all  the  processes  necessary  for  preparing  tissues  and  organs  for  study  according 
to  the  great  groups  of  methods,  like  the  paraffin  and  the  collodion  methods, 
isolation,  and  the  methods  of  study  of  living  and  fresh  material.  It  is  believed 
that  this  training  in  independent  work  is  as  important  as  the  formal  instruction 
in  histology  ;  for  every  investigator  must,  and  every  practitioner  of  either 
human  or  veterinary  medicine,  ought  to  be  able  to  work  out  by  himself  some 
of  the  problems  arising  in  his  study  or  practice. 

An  effort  has  been  made  in  this  course  to  combine  the  excellencies  of  the 
three  great  methods  of  learning,  viz.  :  ( i )  That  of  text-books  and  works  of 
reference  with  recitations  or  quizzes  5(2)  that  of  the  living  teacher  in  lectures, 
and  in  personal  instruction  and  supervision  in  the  laboratory  ;  (3)  finally,  and 
most  important,  that  of  personal  contact  with  the  truths  of  nature  in  actual  work 
where  knowledge  is  gained  at  first  hand. 

If  this  guide  is  looked  over  or  used  by  teachers  in  other  institutions,  we 
shall  deem  it  a  favor  if  any  errors  of  statement  are  pointed  out  to  us,  and  if  sug- 
gestions for  improvement  are  made  by  those  who  have  had  much  experience  in 
conducting  laboratory  courses  with  definite  objects  in  view. 


SIMON  HENRY  GAGE, 

BENJAMIN  FREEMAN  KINGSBURY. 


SEPTEMBER,  1899. 


LITERATURE,  EQUIPMENT  AND  REAGENTS. 


TEXT  BOOK. 

Text-Book  of  Normal  Histology  :  including  an  account  of  the  Develop- 
ment of  the  Tissues  and  of  the  Organs.  By  George  A.  Piersol,  M.  D.  Fifth 
Ed.  Ivippincott  Co. ,  Phila. 


REFERENCE  BOOKS. 

The  following  books  and  journals  will  be  placed  on  the  reference  shelves 
in  the  laboratory,  or  will  be  referred  to  : 

A.  Anatomy  and  Histology. 

Quain's  Elements  of  Anatomy.     Ed.  by  Schafer  &  Thane.     1893. 
Text-Book    of    Histology.     By    Dr.    Philipp    Stohr.     Trans,    by    Dr.    A. 

Schaper. 

Essentials  of  Histology.     By  A.  Schafer. 
Text-Book  of  Histology.     By  Clarkson. 

Manual  of  Histology.     By  S.  Strieker.     Translation.     1872. 
Handbuch  der  Gewebelehre  des  Menschen.     By  A.  Kolliker. 
Lehrbuch  der  Histologie  und    Mikroskopische  Technik.     By  Bohm  and 

and  Davidoff.     1898. 

.  Traite  Technique  d' Histologie.     By  L.  Ranvier. 
Anatomic   du   Systeme    Nerveux   de   1'Homme.     By   A.   van  Gehuchten. 

1897. 

B.  Physiology. 

Text-Book  of  Physiology.     By  M.  Foster. 

An  American  Text-Book  of  Physiology.     Edited  by  W.  H.  Howell. 
Essentials  of  Physiological  Chemistry.     By  Hamersten. 
Text-Book  of  Physiology.     By  E.  A.  Schafer.     1898. 

C.  Embryology. 

Human  Embryology.     By  Minot. 

Text-Book  of  Embryology  :    Man  and  Mammals.     By  O.  Hertwig.    Trans. 

by  Mark. 

Vertebrate  Embryology.     By  Marshall. 
Quain's  Anatomy  :     Part  I,  Vol.  i;  Embryology. 


D.  Journals  and  Periodicals. 

Volumes  of  the  following   may  be  referred  to  and  in  that  case  will  be 

placed  on  the  shelves  : 
Archiv  fur  Mikroskopische  Anatomic. 
Quarterly  Journal  of  Microscopical  Science. 
Journal  of  Anatomy  and  Physiology. 
Journal  of  Physiology. 
American  Journal  of  Physiology. 
Journal  of  the  Royal  Microscopical  Society. 
Anatomischer  Anzeiger. 

Proceedings  of  the  American  Microscopical  Society. 
Journal  of  Morphology. 
Journal  of  Applied  Microscopy. 

E.  Technic. 

In  addition  to  Stohr,  Ranvier,  and  Bohm  &  Davidoff,  consult: 
The  Microtomist's  Vade-mecum.     By  A.  B.  L,ee. 
Methods  of  Pathological  Histology.     By  A.  C.  von  Kahlden. 
Practical  Histology.     By  E.  A.  Schafer. 


PERSONAL  OUTFIT. 

The  student  must  supply  himself  with  the  following  : 

1.  Text-books. 

2.  25  quarto  portfolios  ( 16^x10-^  in. ). 

3.  100  sheets  of  paper,  quarto  size  (8xio^  in.),  for  laboratory  drawings. 

4.  i oo  sheets  of  paper,  ruled  if  preferred,  quarto  size   (8x10^2),  for  descrip- 

tions, etc. 

5.  One  simple  microscope  (tripod  magnifier). 

6.  Two  needle  holders  and  No.  6  needles. 

7.  Fine  forceps,  straight  or  curved. 

8.  Dividers. 

9.  One-half  gross  of  slides;  more  will  be  needed  later. 

10.  Three  slide  boxes. 

11.  Cover  glasses  ;  yz  oz.,  No.  i,  ^  in.  circles;  >^  oz.,  No.  i,  %  in.  circles;  yz 

oz.  oblongs,  No.  i,  23x30  mm. 

12.  One  section  razor  (W.  B.  &  E.,  Phila. ). 

13.  One  or  more  scalpels. 


EQUIPMENT  OF  THE  PERSONAL  LOCKER. 
(Supplied  by  the  Department. ) 

The  personal  locker  contains   the   following,   for  which  the  student   is 
responsible. 

Xylene  balsam,  bottle. 
%%  collodion,  bottle. 

Albumin  fixative  (Mayer's),  homeopathic  vial. 
Eosin,  yi%  aqueous  solution;  bottle  with  pipette. 
Hematoxylin,  large  shell  vial. 
Hematoxylin,  bottle  with  pipette. 
Clearer,  carbol-xylene,  large  shell  vial. 
Clearer,  carbol-xylene,  bottle  with  pipette. 
Picric  alcohol,  bottle. 
Castor  oil,  homeopathic  vial  with  brush. 
Two  Stender  dishes  for  benzin  and  alcohol. 
Carbon  ink,  bottle. 
Pen  and  penholder. 
Glass  jar  for  slides. 
Glass  box  for  cleaning  cover  glasses. 
Two  small  glass  boxes  for  clean  covers. 
Five  medium  shell  vials. 
Three  small  shell  vials. 
Two  watch  glasses. 
Box  of  lens  paper. 
Box  of  vaseline. 
Centering  card. 
Thirty  centimeter  metric  rule. 
Two  slide  trays. 
Two  towels. 
Three  pipettes. 

Five  bottles  for  95%  alcohol,  67%  alcohol,  picrofuchsin,  normal  salt  solu- 
tion and  distilled  water. 


EQUIPMENT  OF  THE  MICROSCOPE  LOCKER. 

Be  careful  not  to  put  any  of  these  in  your  personal  locker,  thereby  caus- 
ing inconvenience  to  others  using  the  same  locker.  Do  not  leave  them  out  on 
the  table. 

A  compound  microscope  with    %  in.  (No.  3),  l/&  in.  (No.  7),  and  1-12  in. 

oil  immersion  objective;  nose  piece;  i  in.  (No.  3  or  4),  and  2  in.  (No. 

i)  ocular;  iris  and  stop  diaphragms;  eye-shade. 

Bottle  of  benzin  for  cleaning  the  immersion  objective. 
Bottle  of  homogeneous  immersion  oil. 
Steel  scale,  marked  in  1-5  mm. 

Glass  micrometer  scale,  ruled  in  i-io  and  i-ioo  mm. 
Slide  of  Pleurosigma  angulatum  (a  diatom ). 
Slide  showing  letters  in  stairs. 


1C 
GENERAL  LABORATORY  REAGENTS. 


Distilled  water. 

Normal  salt  solution. 

67%  alcohol. 

82%  alcohol. 

95%  alcohol. 

Cedar-wood  oil  (thickened). 

Paraffine  for  infiltration. 

Paraffine  for  imbedding. 

Ether-alcohol. 

1/4%  collodion  (thin,  for  infiltrating). 

6%  collodion  (thick,  for  infiltrating). 

8%  collodion  (thick,  for  imbedding). 

%%  collodion. 

Chloroform. 

Clarifier  (castor-xylene). 

Benzin. 

Clearer  (carbol-xylene). 


SPECIAL  LABORATORY  REAGENTS. 

These  will  be  placed  on  the  supply  shelves  when  needed.     There  is  in- 
cluded a  wide  range  of  stains,  fixers  and  solutions. 


INTRODUCTORY. 


The  animal  body  is  made  up  of  different  "organs"  having  spe- 
cial functions.  These  may  be  either  more  or  less  independent  an- 
atomically, or  they  may  be  grouped  together  into  systems.  The 
organs  of  the  body  and  the  parts  of  the  systems  are  composed  of 
different  tissues,  which  in  turn  consist  of  cells  and  special  permanent 
products  of  the  cells — intercellular  substance.  This  analysis  may  be 
set  forth  diagrammatically  as  follows  : 
f  organs  or  \ 

Body.  1  systems  (of  >   Tissues.    < 

I    "organs),    j  |  intercellular  substance. 

The  fundamental  tissues  of  the  body  are  of  four  kinds  : 

A.  Epithelial.     Cells  only  slightly   modified  structurally  and  ar- 

ranged in  layers  or  masses;  intercellular  substance   (cell- 
cement)  small  in  amount  and  undifferentiated. 

B.  Connective   and    Supporting.     Cells   insignificant   in    number, 

slightly  modified  structurally;  intercellular  substance  pre- 
dominant and  variously  modified. 

C.  Muscular.     Cells   specialized    in  structure  for  the  purpose  of 

producing  movement  (i.  e.,  for  contracting  in   one  direc- 
tion). 

D.  Nervous.     Cells  specialized  in  structure  for  the  purpose  of  or- 

iginating,  transmitting,   and    transferring    "nervous    im- 
pulse." 

In   pursuing  the  study  of  Histology  the  following  principles 
are  believed  to  be  the  most  advantageous: 

1.  The  study  of  the  different  tissues  should  precede  the  study 
of  the  organs. 

2.  Every  tissue  and  organ  should  be  studied  fresh  as  far  as 
possible  in  order  to  have  correct  notions  of  the  structural  appear- 
ances unmodified  by  reagents. 

3.  Every  tissue  and  organ  should  be  studied  alive  as  far  as 
possible  in  order  to  see  the  function  and  thus  associate  function  and 
structure. 

4.  Before    the    microscopic   structure   of  an  organ  or  part  is 
studied  t\\z  gross  anatomy  should   be  first  thoroughly  understood. 


12 


Gross  preparations  for  reference  should  be  at  hand  and  constantly 
referred  to  while  the  microscopic  study  is  carried  on. 

5.  In  the  study  of  the  tissues  it  should  be  remembered  that, 
though  at  first  distinct,  in  the  adult  body  there  is  more  or  less 
mingling  of  the  different  tissues;  but  in  this  composite  structure 
there  is  one  tissue  which  from  its  predominance  forms  the  charac- 
teristic feature  of  the  structure.  The  epithelia  are  the  simplest  in 
this  as  in  other  respects.  The  different  organs  likewise  are  not 
isolated  and  independent  units,  but  are  thoroughly  permeated  by 
the  parts  of  the  vascular  and  peripheral  nervous  systems  contained 
within  them. 

In  this  course  the  tissues  and  organs  will  be  studied  in  the 
order  below: 

1.  The  Kpithelia  (including  Kndothelia). 

2.  The    Connecting   and   Supporting  Tissues  and  the 

Skeletal  System. 

3.  Blood  and  Lymph;  i.  e. ,  the  fluids  of  the  body  and 

their  corpuscles. 

4.  The  Muscular  Tissues  and  the  Muscular  System. 

5.  The  Nervous  Tissues  and  the   Peripheral  Nervous 

System. 


WINTER 


6. 

7- 
8. 

9- 
10. 
n. 

12. 


The  Blood  and  Lymph  Vascular  Systems. 

The  Digestive  System. 

The  Respiratory  System. 

The  Urinary  Organs. 

The  Genital  Organs. 

The  Skin  and  Its  Appendages. 

The  Central  Nervous  System. 

The  Organs  of  Special  Sense. 


During  the  fall  term  the  principal  aim  will  be  to  gain  a 
knowledge  of  the  kinds,  structure  and  distribution  of  the  different 
tissues  and  a  working  knowledge .  of  general  histological  methods. 
During  the  winter  term  the  minute  anatomy  of  the  different  organs 
will  be  studied. 

The  connective  and  supporting  and  the  muscular  tissues  are  so 
closely  associated  with  the  organs  and  systems  that  they  principally 


compose  that  the  tissue  and  system  will  be  studied  together.  The 
peripheral  nervous  system,  likewise,  may  best  be  studied  with  the 
nervous  tissues. 

METHOD  OF  LABORATORY  WORK. 

Laboratory  work  is  arranged  as  weekly  exercises,  and  each 
week's  work  is  to  be  completed  before  the  next  succeeding  is  under- 
taken. Mounted  preparations  of  the  various  tissues  and  organs 
taken  up  will  be  studied.  These  preparations  may  be  divided  into 
four  groups:  A,  those  owned  by  the  department  and  assigned  for 
study;  B,  those  prepared  by  the  department  and  simply  to  be 
mounted  by  the  student;  C,  those  in  which  the  fixing,  imbedding 
and  sectioning  are  done  by  the  department  and  the  staining  and 
mounting  by  the  student;  D,  preparations  made  entirely  by  the 
student,  i.  e.,  fixed,  imbedded,  sectioned  and  stained;  and  E,  dem- 
onstrations of  preparations  illustrating  special  features.  With 
all  specimens  prepared  or  partially  prepared  by  the  department 
exact  data  will  be  posted  on  the  bulletin  board  as  to  (a)  the 
mode  of  fixation,  (b)  imbedding  method,  (c)  thickness  of  the 
section,  when  known,  (d)  stains  employed. 

In  the  case  of  preparations  belonging  to  class  D  the  student  is 
expected  to  keep  a  record  of  the  different  steps  and  times.  Since 
there  will  be  slides  owned  by  the  student  (B,  C,  D)  and  by  the  de- 
partment (A),  care  must  be  exercised  in  keeping  them  separate  ; 
those  owned  by  the  student  on  one  slide  tray,  those  of  the  depart- 
ment on  the  other. 

To  gain  a  working  knowledge  of  histological  technic  each  stu- 
dent will  prepare  from  the  beginning  certain  organs  or  tissues,  i.  e., 
fix,  imbed,  section,  stain  and  mount,  imbedding  them  by  the  para- 
fine  and  by  the  collodion  methods. 

LABORATORY    REPORTS. 

Reports  upon  each  tissue  or  system  are  to  be  submitted  when 
the  work  on  that  tissue  or  system  is  finished,  the  reports  and  the 
times  they  are  due  being  posted  on  the  bulletin  board.  These  re- 
ports are  to  consist  in  part  of  drawings  of  the  preparations  studied, 
in  part  of  descriptions  and  such  discussions  as  may  be  specifically 
called  for.  In  the  drawings,  name  all  the  parts  presented  or  at  least 
recognized.  Large  figures  are  preferable  to  small  ones.  Colored 


14 

pencils,  though  not  required,  will  be  found  helpful  in  making  clear 
interpretations.  The  descriptions  should  be  made  out  in  accord- 
ance with  the  following  scheme  : 

Descriptions  for  Laboratory  Reports. 

1.  Name  of  the  specimen. 

2.  Names  of  its  principal  parts. 

3-4.     Constituent  elements  or  tissues  of  each  of  the  main  parts  and 
the  relation  of  the  parts  to  one  another. 

5.  Structures  present  in  the  specimen,  but  not  recognized. 

6.  Distribution  in  the  body. 

7.  Function  of  the  organ  and  of  its  principal  divisions. 

RECITATIONS. 

There  will  be  a  weekly  recitation,  or  quiz,  covering  the  ground 
of  the  lectures  and  the  laboratory  work.  Its  purpose  is  not  only  that 
of  a  weekly  examination,  but  it  is  also  intended  to  unify  the  work 
of  the  week,  bring  out  the  important  features  and  clear  up  difficult 
points.  Weekly  references  to  the  text-book  covering  the  matter  for 
which  the  student  is  held  responsible  will  be  posted,  and  under 
Points  for  Quiz  are  given  the  most  important  features  of  the  work, 
which  the  student  will  be  expected  to  know.  In  the  weekly  exer- 
cises following,  these  are  left  blank  and  should  be  filled  out  from  the 
bulletins  posted. 

EXAMINATIONS. 

There  will  be  a  written  examination  at  the  end  of  each  term, 
covering  the  lectures  and  the  laboratory  work.  In  addition  there 
will  be  a  laboratory  examination  to  test  the  students'  ability  to  recog- 
nize the  different  tissues  and  organs.  In  the  laboratory  examina- 
tion at  the  end  of  the  fall  term,  he  will  be  required  to  recognize  the 
different  tissues  in  the  preparations  given  him.  At  the  end  of  the 
winter  term,  the  identification  of  organs  will  be  expected  of  him. 
In  addition,  at  intervals  during  the  year,  especially  in  the  last  half 
of  the  winter  term,  unlabeled  slides  will  be  assigned  for  study  and 
report.  Also  unknown  tissues  may  be  given  from  which  prepara- 
tions are  to  be  made  and  reported  on  subsequently  as  called  for. 
These  two  methods  will  help  the  student  in  the  recognition  of  the 
different  organs  and  tissues,  and  also  prepare  him  for  the  laboratory 
examinations. 


15 
INTRODUCTORY  AND  TECHNIC. 

LABORATORY  WORK  FOR  THE WEEK. 

I.  Imbedding  tissue.     Go  to  the  delivery  desk    with  two 
small  shell  vials  filled  with  82%  alcohol  from   the  supply  shelf  and 
get  two  pieces  of  tissue  already   fixed   and  hardened  and  now   in 
82%  alcohol.     One  of  these  is  to  be  imbedded  according  to  the  par- 
affine  method  and  sectioned  during  the  week  of  the 
term                          .     The  other  piece  is  to  be  stained  in  toto  in  para- 
carmine   imbedded  in  collodion,  and  sectioned  during  the 

week  of  the  term.  By  means  of  a  ribbon  pin  attach  to  the  cork  of 
each  bottle  a  label  giving  the  name  of  the  animal,  the  tissue  or 
organ,  the  fixer  employed,  the  method  in  which  it  is  to  be  imbedded, 
and  the  date: 

In  order  that  you  accomplish  this  successfully  it  is  necessary 
for  you  to  study  carefully  §§  39-47  ;  50-60.  Keep  a  record  of  the 
steps  and  the  times  of  transfer  to  each  fluid  and  present  it  as  part 
of  the  laboratory  report  on  <V-*-  due  .  (See 

bulletin  board). 

II.  Fixation  of  tissue.     Fill  two    medium  shell  vials  with 
picric-alcohol  and    go  to  the  delivery  desk  for  two  pieces  of  fresh 
tissue,  to  be  fixed  and  hardened,  and  imbedded  according  to  (a)  the 
paraffine  method,   and  (^)  the  collodion  method.     Your  name,  to- 
gether with  the  tissue  you  are  to  receive  and  the  imbedding  method 
to  be  employed  is  posted  on  the  bulletin  board. 

Label  the  specimen,  giving  the  name  of  the  animal,  the  organ 
or  part,  the  fixation  method,  imbedding  method,  and  the  date. 

Keep  a  record  of  the  steps  in  the  process,  and  submit  it  as  part 
of  the  report  for  the  week  of  the  term. 

In  order  that  this  work  be  successfully  pursued,  it  is  necessary 
to  study  carefully  §§  18-21  ;  25  ;  39-47  ;  50-60.  Be  sure  that  you 
understand  the  order  of  succession  of  the  various  steps  and  the  rea- 
son for  them,  and  follow  directions  absolutely.  Fix  in  picric-alcohol 
1-2  days,  place  in  67%  alcohol  1-2  days,  in  82%  alcohol  several  days, 
changing  the  alcohol  to  fresh  three  or  four  times  (§  25).  In 
this  work,  be  as  independent  of  assistance  from  the  instructors  as 
possible. 


i6 

III.  Isolation.     From  the  stock  bottle  on  the  shelves,  fill  a 
small  shell  vial  with  Miiller's  fluid  dissociator  and  go  to  the  delivery 
desk  for  a  small  piece  of  trachea  of  cat  (sheep  or  calf).     Label  it, 
naming  the  animal,  tissue,  and  dissociator.     Leave  the  tissue  in  the 
dissociator  until  you  come  at  the  second  period   (1-2  days)  when  the 
action  of  the  dissociator  will  have  been  sufficient.     For  this  work 
study  carefully  §§  2-7. 

At  your  second  period,  in  accordance  with  the  directions  in 
§§  5-7 >  (a)  examine  the  isolated  cells  unstained,  in  dissociator  ;  (6) 
stain  a  second  preparation  with  methylgreen  and  eosin  (§92,  b), 
and  mount  in  glycerin  ;  (c)  mount  a  third  preparation  of  the  cili- 
ated cells  in  alum-carmine  and  eosin  glycerin  (§  156). 

IV.  Label  the   bottles  of  clearer  ;  fill  and  label  the  bottle  for 
distilled  water  and  those  for  95%  alcohol  and  normal  salt  solution  ; 
prepare  and  label    in   their  respective  bottles   no  c.    c.   of  picro- 
fuchsin  and  90  c.  c.  of  67%  alcohol. 

V.  Make  4  shellac  rings  (cells)  for  ^  in.  cover  glasses  ;  pre- 
pare two  for  y%  in.  covers  (§  106). 

VI.  Read  carefully  §§  i  ;  18-21  ;  64  ;  71-78. 

Review  what  you  have  so  far  done  on  I,  II,  and  III. 
Study  carefully  §§  64-70  ;   79  ;    100-112. 


THE  CELL  AND  THE  TISSUES. 

x-i_^-  LWc*v-tI 

References  :    V  ***** H,    O^- *  >W-*%  ~~  ^  **  ^%,«  VI  |».l  V» 

Points  for  Quiz : 
Laboratory  Report  : 

-x 

Z7z^?  : 

i.  Ovarian  Ova.  Starfish  (Asterias  Forbesii).  Assigned 
for  study.  Picro-acetic  fixation  ;  paraffine  ;  hematoxylin  and 
eosin.  Study,  noting  the  nucleus  (germinal  vesicle),  cell-body 
(vitellus),  nucleolus,  nuclear  membrane,  and  structure  of  the 
nucleus  and  cell-body  (spongioplasm  and  hyaloplasm).  This  prep- 


17 

aration  affords  a  demonstration  that  young  ova  are  single  cells  with 
nucleus  and  cell-body.     Sketch  one  or  more  that  show  typically. 

2.  Ovarian     Ova.      Amblystoma.      Demonstration.      Study, 
recognizing  in   the  young  ova   nucleus   and   cell-body.     Compare 
with  (i). 

3.  Early  blastula  stage.     Amblystoma.     Assigned  for  study. 
At  this  stage  the  fertilized  ovum  has  divided  up  into  a  few  cells, 
arranged  in  a  single  layer  around  a  central  cavity   (blastoccele), — 
comparable  to  a  simple  epithelium.     Make  a  sketch  showing  out- 
lines of  cells  and,  if  possible,  of  nuclei. 

4.  Late  blastula  stage.     Amblystoma.     Assigned  for  study. 
The  cells  at  this  stage  have  greatly  increased  in  number,  are  smaller, 
and  the  wall  bounding  the  cavity  is  of  several  layers  of  cells, — com- 
parable, therefore,  with  a  stratified  epithelium.     Sketch,   showing 
the  outline  of  the  egg,  the  outline  of  the  cavity,  and  for  a  portion  of 
the  wall,  the  outlines  of  the  cells  and  nuclei. 

5.  Medullary    plate    stage.      Amblystoma.      Assigned    for 
study.     At  this  stage  differentiation  is  well  advanced.     The  three 
germ  layers  are  seen  in  the  section  ;  from  the  entoderm  the  noto- 
chord  is  forming  ;  from  the  ectoderm,  the  central  nervous  system  ; 
the  cavity  is  the  alimentary  canal  (archenteron).     Sketch  the  dorsal 
portion,  and  compare  with  the  figure  in  the  text-book. 


TOPICS  FOR  QUIZ— 4th  Week. 

INTRODUCTORY  AND  TECHNIC  ;  THE  CEI,L  AND  THE  TISSUES. 

1.  Histology,  define;  what  is  a  cell,  nucleus,  cell-body;  a  tissue  and  its 
composition,  kinds  of  tissue  ;  intercellular  substance? 

2.  What  is  an  ovum,  and  how  are  the  tissues  derived  from  it? 

3.  What  are  the  ways  of  examining  animal  tissues  with  the  microscope  ? 
What  is  isolation  or  dissociation  ?     Give  examples.     What  is  fixing  and  why 
necessary?     What  is  meant  by   imbedding,  its  aim,  when  necessary?     Name 
three  methods. 

4.  Staining,  what  is  it,  why  advantageous  ;  kinds  of  staining  and  of  stains 
used  in  histology. 

5.  Mounting,  kinds  of  mounting  media  ?     Give  exact  procedure  in  mount- 
ing in  (a)  glycerine  ;  (b)  in  glycerine  jelly  ;  (c)  in  balsam. 


i8 

THE  EPITHELIAL  TISSUES. 

t-t/L 

LABORATORY    WORK    FOR   THE WEEK. 

References: 
Points  for  Quiz  : 
Laboratory  Report  : 

Due: 

Go  to  the  delivery  desk  for  slides  assigned  for  study,  6,  10,  14, 
and  19. 

Prepare  a  clean  slide  with  albumen  fixative  (§  67,  a}  and  go  to 
the  delivery  desk  for  18,  paraffine  section  ;  carry  on  according  to 
§$  67,  a  -70. 

From  the  bottle  on  the  delivery  desk  place  a  small  drop  of 
glycerin  jelly  on  the  center  of  a  clean  slide  and  go  to  the  instructor 
for  9  and  16,  already  stained.  Cover  (§  105). 

Go  to  the  delivery  desk  with  a  clean  slide  for  17,  now  in  thin 
balsam.  Mount  in  balsam  (§109). 

Go  to  the  desk  for  7  ;  in  glycerin  ;  place  a  drop  on  the  slide 
and  cover  it  (§  104). 

Clean  carefully  and  seal  (§  no)  the  preparations  of  ciliated 
cells  from  the  trachea,  made  last  week  (8). 

COLUMNAR    EPITHELIUM. 

6.  Small    intestine.     Cat.     Assigned    for   study.     Transec- 
tion.     Flemming's    fluid    fixation   (§  24)  ;    paraffine   (§  42)  ;  saf- 
franin  stain  (§  79)  ;  sections  10  yu. 

Study  the  epithelium  covering  the  villi,  noting  the  two  kinds 
of  cells  and  the  structure  of  each  ;  the  position  and  shape  of  the 
nucleus  ;  the  striated  border  ;  the  relation  of  the  epithelium  to  the 
underlying  tissue.  Find  a  place  showing  a  surface  section  of  the 
epithelium. 

7.  Epithelial  cells  from  the  small  intestine.    C-o-fc 
Isolated  in  Muller's  fluid  dissociator   (§  4)  ;  now  in  alum-carmine 
and  eosin  glycerin  (§  156). 

)  _   .  £ 


19 

Place  a  small  drop  of  the  glycerin  on  a  slide  and  cover  with  a 
cover- glass.  Search  the  preparation  to  find  (a)  completely  isolated 
cells,  (£)  cells  adhering  together  in  clusters  and  affording  a  surface 
view  of  the  epithelium.  Note  as  before  the  shape  and  structure  of 
the  cells  and  the  shape  and  position  of  the  nucleus.  Try  to  find 
goblet  cells. 

CILIATED    EPITHELIUM. 

8.  Ciliated  cells  from  the  trachea.     Cat.     Two  preparations 
made  last  week. 

Carefully  clean  around  the  cover  glass  and  seal  with  shellac 
(§  no).  Study  the  preparations,  searching  for  completely  isolated 
cells  and  cells  adhering  in  clusters.  Note  the  shape  of  the  cells,  the 
structure  and  position  of  the  nucleus,  the  cilia,  the  shape  of  the 
basal  end.  Compare  the  two  slides,  noting  the  identity  of  structure 
but  different  staining. 

9.  Ciliated  cells  from  the  trachea.   Horse.  Isolated  in  Miil- 
ler's  fluid  dissociator  ;  stained  with  V^c~*^,  ,  now  in    ^U-.i« 
Mount  in  glycerin  jelly  (§  105).     Study  carefully,  noting  the  shape 
of  the  cells,  the  shape  and  position  of  the  nucleus,  the  length  of  the 
cilia.     Compare  with  8. 

10.  Ciliated  epithelium.     Section  of  the  soft  palate.     Cat. 
Assigned  for  study. 

The  nasal  side  will  show  a  so-called  stratified  ciliated  epitheli- 
um. Observe  the  cilia  covering  the  free  ends  of  the  cells  ;  compare 
the,  apparently,  superficial  cells  bearing  the  cilia  with  the  deeper 
cells.  Examine  8  again  to  see  if  you  can  find  any  isolated  cells  of 
the  deeper  layer. 

n.  Living  ciliated  cells.  Go  to  the  desk  with  a  slide  for 
living  ciliated  cells  from  the  oral  epithelium  of  the  frog.  Place 
upon  the  slide  in  a  drop  of  saliva  or  normal  salt  solution  ;  cover. 
Study  the  activity  of  the  ciliated  cells,  trying  to  ascertain  the  move- 
ments of  the  cilia.  This  may  best  be  done  in  cells  in  which  the  cil- 
iary movements  have  considerably  slackened. 

12.  Living  ciliated  cells.    From  the  trachea.     Cat.     Demon- 
stration.    Observe  the  ciliary  movements,  noting  the  rate  and  direc- 
tion of  the  vibrations. 

13.  Ciliated   pavement   epithelium;     From  the   peritoneal 


20 

epithelium  of  an  Amphibian  (Necturus,  Amblystoma.  Frog). 
Demonstration.  Note  the  short  cilia  projecting  up  from  the  flat- 
tened cells  of  the  peritoneal  epithelium. 

PAVEMENT   EPITHELIUM. 

14.  Peritoneal    epithelium    (endothelium).      Mesentery   of 
cat.     Assigned  for  study.     The  fresh  mesentery  was  treated  with  a 
YZ%  aqueous  solution  of  silver  nitrate,  washed  in  water,  exposed  to 
sunlight  until   the  cell- cement   between  the  cells  had  blackened, 
washed  in  water,  stained  in  hematoxylin  to  bring  out  the  nuclei, 
dehydrated,  cleared  and  mounted  in  balsam. 

Study  the  preparation,  noting  the  nuclei  and  cell-outline  as 
indicated  by  the  stained  cell  cement.  Remember  that  the  cells  are 
flat  and  a  single  layer  deep. 

(13).  Ciliated  pavement  (peritonea1!)  epithelium.  Dem- 
onstration. See  13  above. 

STRATIFIED    EPITHELIUM. 

(10).  Stratified  epithelium.  Vertical  section.  Assigned  for 
study.  The  oral  side  of  the  soft  palate  is  covered  with  a  stratified 
epithelium. 

Note  the  shape  of  the  cells  and  nuclei  in  the  deeper  layers  and 
compare  with  the  cells  of  the  superficial  layers,  observing  the  grad- 
ual transition  as  you  pass  from  the  deepest  to  the  superficial  cells. 
Are  intercellular  bridges  to  be  seen  ? 

15.  Stratified     epithelium  ;     surface     cells.       With    the 
finger  nail  gently  scrape  the  inside  of  your  own  cheek   and   mount 
the  scrapings  on  a  slide,   adding  a  drop  of  normal  salt  solution  if 
necessary.     Examine  the  preparation,  noting  the  shape  and  size  of 
the  epithelial  scales,  the  shape  and  size  of  the  nucleus.     The  sur- 
face of  the  cells  may  be  marked  by  ridges  where  neighboring  cells 
have  overlapped.     Can  you  find  groups  of  two  or  more  cells? 

Compare  with  the  superficial  cells  of  10  where  you  see  them  in 
section,  or  on  edge. 

1 6.  Stratified    epithelium ;    deeper    cells.     Isolated   cells 
from  the  deeper  layers  of  the  epithelium  of  the  lip.    W-* 

Miiller's  fluid  dissociator  ;  stained  with  ;  now  in 

Mount    in    glycerin  jelly.      In   studying  this   preparation   find 


21 


cells  in  which  the  intercellular  bridges   (prickles)   are  well  shown, 
noting  as  well  the  shape  and  size  of  the  cells  and  their  nuclei. 

17.  Epidermis.     Human    finger.     Section    slightly   oblique 
with    the   surface.     Fixed  in  <V/^UX^  ;  paraffine  ;  sections 

i  :      yw  ;  stained  with  hematoxylin  ;  now  in  thin  balsam.     Mount 
in  balsam. 

This  is  an  epithelium  in  which  the  scale-like  superficial  cells 
form  a  very  thick  layer  and  their  nuclei  disappear.  In  the  cells  of 
the  deeper  layers  the  nuclei  are  present.  This  preparation  is  to 
illustrate  the  intercellular  bridges  in  a  stratified  epithelium  and  also 
to  show  the  relation  of  the  deepest  cells  to  the  connective  tissue 
upon  which  the  epithelium  rests. 

18.  Follicular  epithelium.     Section  of  the  ovary  of  a  young 
cat.  ;  paraffine;  sections  p. 

Stain  with  hematoxylin  5  minutes,  eosin  15-30  seconds.^  The 
ovary  will  contain  eggs  of  various  ages,  covered  with  a  follicular 
epithelium  of  a  single  layer  of  cells  or  many  layers  of  cells,  depend- 
ing upon  the  maturity  of  the  follicle.  The  follicular  epithelium  in 
the  older  follicles  therefore  is  a  stratified  epithelium  in  which  the 
cells  are  all  cuboidal,  rounded,  or  polyhedral.  Search  the  prepara- 
tion to  find  ova  covered  with  such  an  epithelium  and  note  that  the 
cells  are  approximately  alike,  and  not  as  in  the  ordinary  stratified 
or  squamous  epithelium. 

(10.)  Glandular  epithelium.  Section  of  the  soft  palate.  As- 
signed for  study. 

In  the  substance  of  the  soft  palate  are  contained  numerous 
mucous  glands,  illustrating  glandular  epithelium.  The  tubules  of 
the  glands  are  made  up  of  columnar  cells  with  the  nuclei  in  the 
bases  ;  the  epithelium  in  these  glands  is  therefore  a  simple  columnar 
epithelium. 

19.  Germinal  epithelium.     Section  of  the  ovary  of  a  kitten 
2-3   weeks  old.     Hermann's  fluid    (§  23)  ;  paraffine   (§  42)  ;    iron 
hematoxylin  (§  98)  ;  sections  IJL. 

The  germinal  epithelium  is  the  cuboidal  or  low  cofumnar 
epithelium  upon  the  surface  of  the  young  ovary,  from  which  the  ova 
are  developed.  Examine  the  preparation,  observing  the  surface 
epithelium,  with  here  and  there  a  much  enlarged  cell  with  a  large 
round  nucleus — a  young  ovum. 


22 

THE  CONNECTIVE  AND  SUPPORTING  TISSUES. 

*)^L 

LABORATORY   WORK    FOR    THE--       --WEEK. 

References  : 
Points  for  Quiz  : 
Laboratory  Report : 

Due  : 

Go  to  the  delivery  desk  («;  for  assigned  slides,  20,  22,  27. 

(b)  With  a  slide  prepared  for  a  paraffine  section  (§  67,  a),  26. 

(c)  With  clean  slides  for  28  and  30,  already  stained  and  in  95% 
alcohol  ;  clear  and  mount  in  balsam. 

(d?)  With  clean  slides  for  24  and  25,  now  in  glycerin  ;  mount 
in  glycerin  jelly  (§  105). 

(e)  With  a  clean  slide  for  31  ;  stain  and  mount  as  directed 
below. 

(/")  With  a  clean  slide  for  29  ;  tease  as  directed  below  ;  mount 
in  glycerin  jelly. 

Of)  With  a  clean  slide  for  21,  a  fresh  preparation  ;  treat  as 
directed  below. 

MUCOUS   TISSUE. 

20.  Mucous    tissue.     From 'the    umbilical   cord   of  a    fetal 
mammal.      (Jelly  of  Wharton).     Spread  preparation.     Assigned  for 
study.     Stained  with  hematoxylin  and  eosin. 

In  studying  this  preparation,  note  the  shape  of  the  cells  and 
the  anastomosis  of  their  processes  ;  the  general  homogeneity  of  the 
intercellular  substance.  Compare  with  22. 

AREOLAR   TISSUE. 

r 

21.  Areolar   tissue    (subcutaneous).     Fresh    preparation. 
The  instructor  will  make  for  you  an  artificial  edema  by  injecting 
normal  salt  solution  into  the  subcutaneous  connective  tissue  of  a  kit- 
ten.    This  separates  the  constituents  somewhat  and  enables  one  to 
get  so  thin  a  layer  that  the  different  elements  can  be  easily  made  out. 


23 

Cut  out  a  small  piece  with  the  scissors  and  spread  it  out  in  a 
thin  layer  on  the  slide,  using  a  dry  slide  and  new  needles.  Draw 
the  edges  away  from  the  middle,  and  if  they  are  dry  enough  they 
will  stick  to  the  slide  and  hold  the  film  outspread.  It  will  probably 
be  necessary  to  absorb  some  of  the  liquid  by  means  of  lens  paper  be- 
fore you  can  make  the  edges  stick.  Do  not  allow  the  middle  to  dry. 
Place  on  the  middle  a  drop  of  normal  salt  solution  and  a  cover-glass. 

Examine  the  preparation  carefully,  recognizing  (#)  the  white 
fibers,  in  wavy  bundles  ;  (£)  the  yellow  elastic  fibers,  straight  and 
single  ;  if  possible  find  a  place  where  they  anastomose  (see,  how- 
ever, below)  ;  (»  the  connective  tissue  cells  ;  can  you  find  more 
than  one  kind?  (See  22). 

Action  of  Reagents.  Remove  the  cover  glass  and  place  upon 
the  preparation  a  drop  of  2%  acetic  acid.  Note  that  it  makes  some 
of  the  fibers  disappear,  the  white  fibers,  while  others,  the  yellow 
elastic,  are  sharply  brought  out.  It  is  now  possible  to  observe  the 
course,  anastomosing  and  branching  of  the  yellow  fibres.  Note 
also  that  the  nuclei  of  the  connective  tissue  cells  are  more  sharply 
defined. 

22.  Subcutaneous  areolar  tissue.     Stained   preparation. 
Assigned    for   study.     Tissue   spread    on   the    slide   by   means  of 
needles,    as  in   21  ;  stained  with  hematoxylin  and  erythrosin   (or 
eosin)  (§  79). 

Study  the  preparation  carefully,  recognizing  again  the  white 
and  yellow  fibers  and  the  cells,  of  which  find  if  possible  three 
kinds  (text-book). 

23.  Adipose    tissue.     Demonstration.     Spread    preparation. 
Stained  with  hematoxylin  and  eosin  (§  79). 

In  this  preparation  of  subcutaneous  adipose  tissue  note  (a)  the 
lobular  grouping  of  the  fat  cells,  (^)  the  blood  vessels  and  the  rela- 
tion of  the  fat  cells  to  the  capillary  network,  (<:)  the  structure  of 
the  individual  fat  cells,  the  fat  globule,  the  nucleus  with  the  proto- 
plasm surrounding  it. 

WHITE   FIBROUS  TISSUE. 

24.  Tendon.     From  the  tail  of  a  mouse.     Tendons  pulled 
out,  spread  out  flat  by  means  of  a  needle  and  stained  15-20  minutes 


24 

with  Khrlich's  acid  hematoxylin  (§  81),  now  in  glycerin.   Mount  in 
glycerin  jelly  (§  105). 

To  show  the  tendon  cells  (connective  tissue  cells).  Note  their 
arrangement  in  parallel  rows  between  the  tendon  fibers,  their  shape 
in  relation  to  each  other,  the  position  of  the  nucleus  in  the  cell  in 
relation  to  the  nucleus  of  the  contiguous  cell.  Consult  also  26  for 
the  true  shape  of  the  cells. 

25.  Tendon.  From  the  tail  of  a  mouse.  Silvered  to  show  neg- 
ative images  of  the  tendon  cells  (§  147).  The  tendons  were  pulled 
out  and  treated  for  a  few  minutes  with  a  solution  of  silver  nitrate, 
washed  in  water,  exposed  to  the  light,  washed  in  water  ;  now  in 
glycerin.  Mount  in  glycerin-jelly.  The  places  occupied  by  the 
cells  are  white,  while  the  cement  substance  was  colored  brown  by 
the  silver  nitrate  on  exposure  to  the  light.  Note  the  outlines  of  the 
cells  and  compare  with  the  positive  images  of  24. 

26.  Tendon.     Transection   of  tendon,  biceps  of  a   new-born 
puppy.     Miiller's  fluid  (§  29)  ;  paraffin  ;  sections  yw. 

Stain  30  minutes  with  hematoxylin,  15  seconds  with  picro- 
fuchsin.  Mount  in  balsam.  Note  the  relation  of  tendon-cells  and 
fibers  as  shown  in  transection  and  compare  with  24  and  25.  Note 
also  the  staining  reactions  of  the  white  fibrous  tissue  with  picro- 
fuchsin  in  comparison  with  the  transected  muscle  fibers  (central), 
and  the  elastic  tissue  in  28. 

27.  Cornea.     Cat.      Assigned  for  study.     Free-hand  sections. 
The  cornea  silvered  (§  147),  free-hand  sections  cut  from  the  ental 
surface  and  mounted  in  glycerin  jelly.  v< 

To  show  the  negative  images  of  the  cells  of  dense  connective 
tissue  (i.  e.,  the  cornea).  Note  the  shape  of  the  cell  spaces,  and  the 
anastomosing  of  the  processes  ;  compare  with  the  similar  preparation 
showing  the  cell  spaces  in  tendon,  25. 

YELLOW   ELASTIC   TISSUE. 

28.  Ligamentum  nuchae.     Horse.     Transection.  ; 
paraffin  ;  sections               yw.     Stained  with  hematoxylin   and   picro- 
fuchsin,  now  in  95%  alcohol.     Clear  and  mount  in  balsam. 

Note  the  yellow  elastic  fibers,  their  shape  and  size  in  transec- 
tion, the  white  connective  tissue  in  between  them,  and  the  nucleus 


25 

of  an  occasional  connective  tissue  cell.     What  is  the  staining  reac- 
tion with  picrofuchsin  ?     Compare  with  26. 

29.  Ligamentum     nuchae.      Horse.      Teased    preparation. 
With  your  needles  tease  out  very  carefully   a  small  piece  of  liga- 
mentum  nuchae  obtained  from  the  instructor.     Remember  that  the 
course  of  the  elastic  fibers  is  longitudinal,  and  therefore  tease  by 
pulling  the  fibers  to  the  side,  trying  to  keep  them  parallel.     Tease 
carefully  and  thoroughly.      Mount  in  glycerin  jelly. 

Note  the  anastomosing  and  branching  of  the  fibers  ;  are  trans- 
verse markings  to  be  detected  ? 

PIGMENT   CELLS. 

30.  Pigmented  pia.     Sheep.     From  the  frontal  region  of  the 
brain.     Now    in   95%    alcohol  ;  no   staining   required.     Clear   and 
mount  in  balsam. 

Note  the  shape  and  anastomosing  of  the  pigment  cells,  the  pig- 
ment granules,  and  the  clear  space  occupied  by  the  nucleus  of  the 
cell. 

31.  Pigmented     peritoneum.     Necturus.      Picric    alcohol  ; 
peritoneum   removed  and   now  in   82%  alcohol.     Stain  with  hema- 
toxylin  10  minutes,  eosin    15   seconds   (§  94).     Mount  in   balsam. 
Since  this  preparation  is  not  fixed  to  the  slide,  care  must  be  taken 
in  changing  fluids  (§  65,  a). 

Note  the  shape,  the  nucleus,  the  pigment  granules,  and  the 
state  of  expansion  or  contraction  of  the  pigmented  connective  tissue 
cells.  Does  the  preparation  show  cells  well  expanded  and  cells 
quite  contracted? 

TOPICS  FOR  QUIZ— 6th  Week. 

THE  CONNECTIVE  AND  SUPPORTING  TISSUES. 

1.  General  character  of  all  the  connective  tissues  ?    Give  the  kinds. 

2.  Mucous  tissues,  character  and  distribution  ? 

3.  Aroelar  tissue,  character  and  distribution  ? 

4.  Fibrous  tissue,  kinds  and  distribution  of  each  variety  ? 

5.  Special  cells  found  in  connective  tissue.     Where  are  they  found  in  man 
and  in  some  domestic  animal  ? 

6.  Compare  the  cellular  and  the  intertellular  or  ground  substance. 

7.  What  is  the  effect  of  reagents  on  the  cells  and  on  the  ground  substance  ? 

8.  From  what  germ  layer  is  connective  tissue  developed,  and  in  what  form 
does  it  first  appear  ? 


26 

CONNECTIVE  AND  SUPPORTING  TISSUE. 

LABORATORY    WORK    FOR    THE  ______  WEEK. 

References  : 
Points  for  Quiz  : 

Laboratory  Report: 

-. 
Due  :    WWu  . 

Go  to  the  delivery  desk  («)  for  35,  assigned  for  study  ;  (b)  with 
a  slide  prepared  for  a  paraffin  section,  32  ;  (c)  with  a  clean  slide  for 
collodion  sections,  37,  38,  39  ;  fasten  to  the  slide  according  to  §  66  ; 
place  in  benzin. 

While  the  above  preparations  are  being  carried  on  (§§  68-70)  go 
to  the  desk  for  33  and  34,  from  which  free-hand  sections  of  the  car- 
tilage are  to  be  cut,  stained  and  mounted  as  directed  below.  Return 
the  tissue  to  the  desk  when  you  have  finished  with  it. 

During  the  week  you  will  make  preparation  36,  in  accordance 
with  the  directions,  §§  133-134.  Read  them  carefully  in  advance. 

CARTILAGE. 


32.  Embryonal  cartilage.       ^-Js-^o    ~*~-  Transection  of 

.  Vr<A^iXo.         .  stained  in  toto  (§  74,  c,  §  86)  in 
paraffin;  sections     "1      JA.     Mount  in  balsam  (benzin, 
clearer,  balsam). 

The  skull  should  be  studied.  It  is  at  this  stage  cartilaginous, 
and  the  cartilage  in  an  embryonic  stage  ;  note  the  cells  with  their 
nuclei  and  cell  -bodies  ;  the  small  amount  of  matrix  deposited  be- 
tween them,  and  compare  with  adult  cartilage  in  33  and  38.  What 
would  be  the  structure  of  the  cartilage  at  an  earlier  stage  of  develop- 
ment? 

33.  Hyalin    cartilage.     Necturus    (Amphibian).     You    will 
receive  one  of  the  following  from  which  to  cut  free-hand  sections  of 
cartilage,  (#)  head  of  the  humerus  ;    (b)  head  of  the  femur  ;    (f)   a 
branchial  arch  ;    (d)   part  of  the  hyoid  arch  ;    (e)  the  pectoral  arch  ; 
(/")  the  pelvic  arch. 


27 

Cut  the  sections  very  carefully  and  as  thin  as  possible.  Place 
6-10  good  sections  in  an  alum  solution  (^4%)  in  a  watch-glass  for 
5-10  minutes  ;  place  3-4  sections  on  a  slide  in  a  drop  of  normal  salt 
solution  ;  cover  and  examine,  studying  the  character  of  the  cells 
arid  their  nuclei,  their  grouping,  the  character  of  the  matrix. 

After  the  5-10  minutes,  drain  off  the  alum  solution  from  the  sec- 
tions in  the  watch-glass.  Mount  one-half  of  them  directly  in  gly- 
cerin jelly;  the  remainder,  stain  in  hematoxylin  for  5-10  minutes, 
wash  in  water,  and  mount  in  glycerin  jelly.  These  two  prepara- 
tions are  to  be  studied  in  connection  with  the  sections  in  normal  salt 
solution,  for  the  structure  of  hyalin  cartilage. 

(38).  Hyalin  cartilage.  Mammal.  For  this,  study  38, 
noting  the  structure  of  the  cartilage,  the  grouping  of  the  cells,  their 
shape  and  arrangement,  in  the  deeper  portion  and  at  the  articular 
surface,  and  the  corresponding  difference  in  the  density  of  the  matrix. 
Compare  with  33. 

34.  Elastic  cartilage.     From  the  ear  of  the  ox  (or  horse). 
Picric  alcohol  fixation.      Make  free-hand  sections,  cutting  them  as 

thin  as  possible.     Place  three  or  four  of  the  thinnest  in  water  for  ,5 

2.O     .  .  .     1-o   rxct>o\^S 

minutes,  stain  in  hematoxylin  *•§,  minutes,  picrofuchsm  f-a  minutes  ; 

dehydrate,  clear,  and  mount  in  balsam.  Study  carefully,  noting  the 
areas  of  hyalin  matrix  surrounding  the  cells  and  the  reticulum  of 
elastic  tissue.  Compare  with  sections  of  hyalin  cartilage,  33,  38, 
and  contrast  with  the  ligamentum  nuchae  (28).  Note  again  the 
different  staining  reaction  of  the  elastic  fibers  of  the  cartilage  and  the 
white  fibrous  tissue  surrounding  the  cartilage. 

35.  Fibro-cartilage.  From  the  inter-vertebral  disc. 
Assigned  for  study.                       ;  ;  stained  with 

There  are  shown  the  interlacing  bundles  of  white  fibrous  tissue  with 
the  cartilage  cells  surrounded  by  hyalin  matrix  scattered  through 
the  tissue.  Compare  with  the  elastic  cartilage,  34,  and  in  this  com- 
parison recall  the  staining  reaction  of  the  white  and  the  elastic  fibers 
(26,  28). 

BONE. 

36.  Dry  bone.    Homo.    Transection  of  femur.    To  be  ground 
down  to  a  thin  section  and  mounted  in  accordance  with  the  special 
directions,  §§  133-134. 


28 

Study  the  preparation  very  carefully,  recognizing  (#)  the  Ha- 
version  systems,  (£)  the  interstitial  lamellae.  In  the  Haversian 
systems,  note  the  Haversian  canal,  the  concentric  lamellae  of  bone, 
the  lacunae  (cavities  occupied  during  life  by  the  bone  cells),  the 
canaliculi.  Understand  from  the  development  of  bone,  the  meaning 
of  the  concentric  arrangement  constituting  an  Haversian  system. 
In  the  interstitial  bone,  observe  the  lacunae  and  canaliculi.  What 
is  the  meaning  of  interstitial  lamellae  ? 

If  desired,  a  tangential  section  of  femur  may  be  prepared  to 
show  the  lacunae  from  another  aspect,  and  thus  gain  a  better  idea 
of  their  real  shape. 

37.  Developing  bone.     Longisection  of  the   hand   (or  foot) 
of  a  fetal  mammaL     Fixed  in  ^iurv*(M»  collodion;  sections    1^     yu. 
Stain  with  hematoxylin  minutes  ;  picrofuchsin  seconds. 

This  illustrates  endochondral  ossification  (early  stage)  and  the 
cartilaginous  stage  of  the  primary  (endochondral)  bones — in  this 
case  the  phalanges  and  carpal  (or  tarsal)  bones. 

In  at  least  one  of  the  phalanges  that  is  cut  longitudinally  the 
early  stages  of  ossification  may  be  seen.  Study  it  carefully,  noting 
the  areolation  of  the  cartilage,  identifying  if  possible  the  osteo- 
blasts  and  osteoclasts  ;  if  the  section  is  in  the  right  plane  the  in- 
growth of  periosteum  may  be  seen.  If  the  process  of  ossification  is 
far  enough  advanced,  note  the  formation  of  the  primary  marrow 
cavity  and  the  lamellae  of  spongy  bone.  The  later  processes  of  os- 
sification are  shown  in  28. 

In  addition  the  preparation  illustrates  :  (#)  true  or  diarthrodial 
articulations,  (£)  capsular  ligaments,  (<:)  muscle  and  tendon,  and 
(d)  developing  hair. 

38.  Developing   bone.^^A?V^^<^ongitudmal   section   of 
the  head  of  a  long  bone  VXAI\V\&T(^  picric  alcohol  ;  decalcified  in 
nitric  acid  decalcifier  (§§  131-132)  ;  collodion  ;    sections       50      /*. 
Stain    in    hematoxylin    30-45    minutes,    picrofuchsin    l/2-i    minute. 
Mount  in  balsam. 

This  preparation  will  illustrate  (a)  the  formation  of  endochon- 
dral bone,  (£)  the  structure  of  the  epiphyses  and  the  longitudinal 
growth  of  a  long  bone.  Recognize  the  following  features  :  (i)  the 
center  of  ossification  of  the  epiphysis,  (2)  the  center  of  ossification 
for  the  shaft,  (3)  the  marrow,  (4)  the  bone,  (5)  cartilage,  (6)  peri- 


29 

osteum.  Study  carefully  the  centers  of  ossification  in  the  epiphysis 
and  shaft,  so  as  to  understand  the  dissolution  of  the  cartilage  (de- 
generation of  the  cells,  absorption  of  the  matrix)  and  the  deposit  of 
bone.  How  is  spongy  bone  converted  into  dense  bone  ?  Study  the 
structure  of  the  periosteum,  recognizing  the  two  layers  ;  understand 
the  part  it  plays  in  the  processes  of  ossification.  In  addition,  study 
the  preparation  for  the  structure  of  cartilage,  articular  cartilage,  the 
general  structure  and  articulation  of  a  long  bone^ 

39.  Transaction  of  a  long  bone.  VvA?V*^  JUA^/WMJ^-O  Picric 
alcohol;  decalcified  in  nitric  acid  decalcifier  (§  131)  ;  collodion  ;. 
sections  CLi*  //.  Stain  in  hematoxylin  bO  minutes,  0 


seconds.     Mount  in  balsam. 

Study,  noting  (a)  the  marrow  with  its  nutrient  artery,  (£)  the 
structure  of  the  bone,  (c)  the  structure  of  the  periosteum.  Com- 
pare the  structure  of  the  decalcified  bone  with  the  dry  preparation 
of  bone,  36.  Recognize  the  identity  of  structure  in  the  two  prepar- 
ations so  differently  prepared. 


TOPICS  FOR  QUIZ— 7th  Week. 

THE  CONNECTIVE  AND  SUPPORTING  TISSUES  CONTINUED. 

9.     Cartilage,  general  character  ;  varieties,  and  the  situation  of  each? 
10.     Embryonal  cartilage.     When  found  and  where  situated? 
ir.     Hyalin  cartilage,  character  and  distribution? 

12.  Elastic  cartilage,  character  and  distribution? 

13.  Fibro-cartilage,  character  and  distribution? 

14.  Bone,  the  varieties,  structure  and  situation  ? 

15.  Development  of  bone.     Be  ready  to  give  the   steps  as  described   in 
Piersol. 

1 6.  Centers  of  ossification  ? 

17.  Compare  the  intercellular  or  ground  substance  of  bone  and  cartilage 
with  that  of  the  other  connective  tissues. 


30 
BLOOD  AND  LYMPH. 

LABORATORY    WORK    FOR   THE WEEK. 

D    f 

References  : 
Points  for  Quiz  : 
Laboratory  Report  : 

Due: 

Go  to  the  delivery  desk  with  two  clean  slides  for  45  and  46  now 
in  balsam  ;  place  a  small  drop  of  the  balsam  on  the  slide,  and  cover. 

Obtain  from  the  desk  48,  spread  on  a  cover-glass  ;  stain  as 
directed  below  and  mount  in  balsam. 

To  economize  time,  next  prepare  42  and  43. 

Obtain  47  at  your  second  period. 

HUMAN  BLOOD. 

40.  Fresh  blood.     Flame  a  needle  and  after  it  is  cool,  prick 
the  finger  near  the  nail-bed  (best)  or  the  side  of  the  finger  ;  wind  a 
handkerchief  around  the  base  of  the  finger  and  press  the  finger  with 
the  other  hand  ;  a  drop  of  blood  will  be  obtained.     Or,  you  may  dis- 
infect the  needle  of  a  haemospath  and  with  it  obtain  the  blood  more 
quickly  and  easily  (§  120).     Touch  a  -cover-glass  to  the  large  drop 
so  obtained  and  place  it  upon  a  slide,  blood  side  down.     The  drop 
of  blood  should   be  large  enough  to  easily  fill  the  space  under  a 
cover-glass.     Seal  the  preparation  at  once  with  castor  oil. 

Study  the  corpuscles  carefully,  noting  the  form  as  determined 
by  observing  the  face,  profile  and  oblique  views  ;  the  corpuscles  can 
be  made  to  turn  over  by  pressing  gently  on  one  edge  of  the  cover- 
glass.  Observe  that  most  of  the  corpuscles  are  arranged  in  rolls  or 
rouleaux.  Here  and  there  may  be  seen  a  leucocyte  or  white  blood 
corpuscle. 

41.  Action  of  reagents.     Obtain  a  large  drop  of  blood  as  in 
40  ;  ascertain  the  effect  upon  fresh  blood  of  four  reagents  as  given 
below.     Since  this  will  take  some  time  and  needs  fresh  blood  it  may 
be  necessary  to  prick  the  finger  more  than  once. 

(a)  Normal  salt  solution.  Place  upon  a  clean  slide  a  small 
drop  of  fresh  blood  and  add  to  it  a  small  drop  of  normal  salt  solu- 


tion  ;  apply  a  cover  glass.  Is  any  change  observable  ?  If  so,  what  ? 
Is  the  fluid  truly  normal  ? 

(6)  Distilled  water .  Place  upon  a  clean  slide  a  drop  of  fresh 
blood  and  place  beside  it  a  small  drop  of  distilled  water  ;  cover. 
What  is  the  effect  of  the  water  upon  the  corpuscles  ?  Where  the 
blood  and  water  meet  observe  the  changes  ;  find  some  corpuscles 
entirely  in  the  water.  What  is  the  meaning  of  the  change  ? 

(c)  2  %  salt  solution.  Place  a  small  drop  of  blood  upon  a  slide, 
cover,  and  at  the  side  ot  the  cover  glass  place  a  drop  of  a  2%  solu- 
tion of  common  salt.  What  is  the  effect  of  the  solution  upon  the 
corpuscles  with  which  it  comes  in  contact  ?  Meaning  ? 

(X)  2  %  acetic  acid.  Make  another  preparation  of  fresh  blood 
and  at  the  side  of  the  cover  glass  place  a  drop  of  a  2%  solution  of 
acetic  acid.  Note  the  effect  of  the  solution  upon  the  red  corpuscles 
and  compare  with  the  action  of  distilled  water.  What  is  the  effect 
of  the  solution  upon  the  white  corpuscles?  What  is  the  meaning 
of  these  experiments  in  connection  with  the  structure  of  the  red 
corpuscles  ? 

42.  Dry  preparation.  With  a  piece  of  lens  paper  wet  with 
95%  alcohol  wipe  off  the  finger  at  the  point  where  it  is  intended  to 
obtain  blood  ;  prick  the  finger  as  in  40,  and  as  directed  in  §  121, 
( i ) ,  prepare  six  or  more  blood  films  upon  clean  covers  ;  allow  two 
of  them  to  dry  and  place  the  others  in  a  watch  glass  of  ether- alcohol 

c  A^XXVt 

for  43. 

Allow  the  two  films  saved  out  to  dry  thoroughly,  then  warm 
gently  and  mount  the  best  (or  both)  film  side  down  upon  a  shellac 
ring  prepared  at  some  previous  time  (§  103,  a).  Select  a  place  where 
the  film  is  thin  and  even  to  study  the  appearance  of  the  dried  cor- 
puscles. Choose  several  corpuscles  that  are  not  distorted  and  with 
the  ocular  micrometer  for  which  you  determined  the  valuation 
measure  their  diameter  in  microns.  Compare  with  the  size  given 
in  the  text -book. 

13.  Stained  preparations.  (§§121-123).  Allow  the  prep- 
arations that  were  placed  in  ether-alcohol  to  fix  for  one  hour  or 
longer,  remove  them  and  allow  them  to  dry  for  a  few  minutes,  when 
they  may  be  stained  immediately  or  later  (at  your  second  period). 
Stain  the  blood  films  in  two  ways  :  (a)  with  eosin  and  hematoxylin 


32 

(§  124)  ;  rinse  in  water  and  dry.  Mount  in  balsam  without  clear- 
ing (§  IO9)>  (^)  with  Ehrlich?  s  triacid  mixture  (§  125). 

In  the  first  preparations  the  red  corpuscles  will  be  red  or  pink, 
in  the  second  yellow  or  orange.  Compare  with  42  as  to  shape  and 

size. 

44.  Blood-plates.     Demonstration.      The  tip  of  the   finger 

was  carefully  cleaned  with  95%  alcohol,  the  finger  pricked,  a  drop  of 
i%  osmic  acid  was  placed  over  the  puncture  and  the  blood  squeezed 
out  into  the  drop  of  osmic  acid,  which  was  transferred  to  a  slide, 
covered,  and  sealed  with  castor  oil. 

Note  the  appearance  and  size  of  the  blood-plates  in  comparison 
with  the  red  corpuscles. 

AMPHIBIAN    BLOOD. 

45.  Blood  of  Necturus.     A  Necturus  was  pithed,  the  gills 
cut,  and  the  drops  of  blood  caught  in  a  vial  of  i%  osmic  acid.     It 
was  allowed  to  fix  in  this  1-2  hours,  washed  in  water,  changed  sev- 
eral times,  washed  in  67%  alcohol,  stained  several  hours  in  paracar- 
mine  (§  86),  washed  in  82%  alcohol,  95%  alcohol,  absolute  alcohol, 
xylene  and  xylene  balsam.     Cover  a  drop  of  the  balsam  containing 
the  corpuscles. 

Note  the  shape  of  the  corpuscles,  their  nuclei,  size  as  compared 
with  human  blood  and  frog's  blood  (42,  46). 

46.  Blood  of  frog.     Prepared  as  was  45.      Mount  a  drop  of 
the  balsam  containing  the  corpuscles.     Compare  with  42,  45. 

LAMPREY  BLOOD. 

47.  Place  a  drop  of  the  blood  of  the  lamprey  upon  the  center 
of  a  slide,  apply  a  cover-glass,  and  seal  with  castor  oil. 

Study  carefully  the  form  of  the  red  corpuscles  and  compare  with 
human  (mammalian)  blood  as  to  form  and  size.  Do  they  arrange 
themselves  in  rouleaux  ?  Observe  the  occurrence  of  leucocytes, 
their  number  and  size. 

Carefully  wipe  away  the  castor  oil,  lift  the  cover  a  little,  and 
place  under  the  edge  a  small  drop  of  2%  acetic  acid.  What  is  the 
effect  ?  Compare  with  the  effect  of  acetic  acid  upon  human  blood. 

What  differences  are  there  between  the  red  corpuscles  of  mam- 
malia, amphibia,  and  the  lamprey  ? 


33 

DEVELOPING   RED   BI,OOD    CORPUSCLES. 

48.     Smear  preparation  of  red  marrow. 

A  drop  or  red  marrow  was  spread  upon  a  cover-glass  as  a  thin  film 
(§  12 1 )  ;  fixed  in  a  saturated  solution  of  mercuric  chlorid  (§  26) 
15  minutes  or  so,  and  placed  in  67%  alcohol.  Rinse  off  the  alcohol 
with  distilled  water  and  stain  with  Bhrlich's  triacid  mixture  (§  125) 

ifWi 

for    10-15   minutes,   rinse  with  distilled  water,   dry  and  mount  in    - 
balsam. 

In  this  preparation  find  («)  marrow  cells,  medium  sized  cells 
with  a  large  nucleus  and  scanty  protoplasm,  resembling  leucocytes 
somewhat  ;  (^)  giant  cells,  with  one  or  many  nuclei  ;  (e)  cells  with 
deeply  staining  nuclei  and  more  or  less  hemoglobin  in  the  cell  body 
(stained  orange), — erythroblasts  ;  (d)  finally,  red  blood  corpuscles, 
non-nucleated.  Find  as  many  forms  of  erythroblasts  as  possible, 
and  compare,  with  the  red  corpuscles. 


TOPICS  FOR  QUIZ— 8th  Week. 

THE   BlyOOD   AND   THE   IvYMPH. 

1.  What  and  where  is  blood,  (a)  plasma  or  intercellular  substance;  (b) 
corpuscles  ? 

2.  What  and  where  is  lymph,  (a)  plasma  ;  (b)  corpuscles? 

3.  Character  of  the  corpuscles  in  the  vertebrates  ? 

4.  Size  of  the  corpuscles  in  man,  horse,  ox,  dog,  necturus? 

5.  Methods  of  study  ;  moist,  dry  and  stained  preparations. 

6.  Compare    the   blood    corpuscles  of  man,   rabbit,  camel,  lamprey  and 
necturus. 

7.  Action  of  reagents  on  the  corpuscles? 

8.  Development  of  the  corpuscles  in  the  embryo  and  in  the  adult? 


34 
BLOOD  AND  LYMPH. 

LABORATORY    WORK    FOR    THE WEEK. 

References  : 
Points  for  Quiz : 

T         L  ,  T>     z.  U    l>    ,     U    V,U     1 

Laboratory  Report: 

Due  : 

Go  to  the  delivery  desk  for  51;  stain  as  directed  below  and 
mount  in  balsam.  Next  prepare  53.  Make  the  remaining  prepara- 
tions in  order. 

LEUCOCYTES. 

49.  Amoeboid    movement.     Amphibian  leucocytes.     Place 
upon  the  slide  a  drop  of  frog's  blood  ;  apply  a  cover  glass  at  once 
and  seal  the  preparation  with  castor  oil. 

Search  the  preparation  until  a  leucocyte  is  found  undergoing 
amoeboid  movement.  Keep  it  under  observation  for  some  time,  ob- 
serving the  changes  of  form  and  mode  of  progression.  Make  sev- 
eral sketches  at  intervals  of  2-3  minutes  showing  the  changes. 

50.  Amoeboid    movement.      Human    leucocytes.     Place   a 
drop  of  fresh  human  blood  upon  a  slide,  cover  quickly  and  seal  with 
castor  oil.     Ten  or  fifteen  minutes  after  the  preparation  was  made 
examine  it,  find  a  leucocyte  in  amoeboid  movement.     Study  it  care- 
fully and  make  several  sketches  at  intervals,  as  in  49. 

51.  Ingestion  of  foreign  particles.     Leucocytes  of  rat.     A 
solution   containing  particles   of  lampblack    (§  157)    was    injected 
into  the  abdominal   cavity   of  a  rat  ;  the  following  day  the  rat  was 
killed  and  smears  were  made  of  the   abdominal   lymph.     Stain  for 
10  minutes  with  hematoxylin  (§  124).     Mount  in  balsam. 

Study  the  preparation,  noting  that  the  particles  of  lampblack 
have  been  taken  up  or  ingested  by  the  leucocytes  of  the  lymph. 
Consider,  in  the  light  of  49  and  50,  how  this  ingestion  took  place. 

(43)  Forms  of  leucocytes.  Study  the  preparations  43,  (a) 
and  (£),  searching  carefully  for  different  kinds  of  leucocytes.  The 
following  types  will  probably  be  found  :  (a)  small  lymphocytes, 


35 

small  leucocytes  with  a  large  rouud  nucleus  and  scanty  protoplasm, 
(£)  large  lymphocytes,  large  leucocytes  with  a  large,  clear  nucleus 
and  rather  scanty  protoplasm,  (V)  polymorphonudear  neutrophiles, 
medium-sized  leucocytes  with  irregular,  horse-shoe  shaped  nucleus 
or  several  nuclei,  the  protoplasm  granular,  and  (d)  leucocytes  with 
the  nucleus  irregular  or  in  two  or  more  parts,  protoplasm  containing 
large  granules  that  stain  intensely  with  eosin  (or  other  acid  staining 
principle),  hence  they  are  called  "eosinoph ties" .  (V)  will  be  most 
abundant,  (a)  and  (b)  less  abundant,  (d)  least  abundant.  It  is 
generally  believed  that  (#),  (£),  (V),  and  (d)  are  stages  in  the 
growth  of  a  leucocyte  from  youth  to  old  age.  Consult 

FIBRIN. 

52.  Upon  a  clean  cover  glass  place  a  large  drop  of  fresh  human 
blood   and  cover  it  with  another  cover  glass,  taking  care  that  the 
two  covers  are  a  little  eccentric  to  each  other.     Place  the  two  covers 
so  prepared  in   a  moist  chamber  (on  the  table)  for  15  minutes  or 
longer.     Transfer  the  two  covers  to  a  slide,  flood  them  with  water 
and  carefully  separate  them.     Wash  the  film  side  of  each  very  care- 
fully with  water  by  means  of  a  pipette  to  remove  the  red  corpuscles. 
Stain  the  film  side  with  eosin  or  erythrosin  for  5  minutes,  drain  off 
the  stain  and  dry  without  washing.     When  dry  mount  the  better  of 
the  two  preparations  upon  a  shellac  ring  film  side  down. 

Observe  the  network   of  the   fibrin  coagulum   adherent  to  the 
cover. 

BLOOD   CRYSTALS. 

53.  Hemoglobin.     Place  upon  a  slide  a  small  drop  of  mam- 
malian blood  (rat,  cat,  or  human)  ;  place  beside  it  a  small  drop  of  a 
10%  aqueous  solution  of  pyrogallic  acid  ;  cover  both  drops  with  a 
cover-glass  and  seal  carefully  with  castor  oil.     Label  the  slide  so 
prepared  with  your  name  and  place  it  upon  the  window  sill.     At  the 
second  period  (i.  e.,  after  one  or  two  days),  examine  it  for  crystals 
of  hemoglobin.     These  will  be  found  near  the  line  where  the  drops 
of  blood  and  pyrogallic  acid  met.     Observe  their  color,  shape  and 
arrangement. 

54.  Oxy-hemoglobin.     Blood  of  Nedurus.     Demonstration. 
A  plentiful  supply  of  Nedurus  blood  was  placed  upon  a  slide,  mixed 


36 

with  a  drop  of  2%  aq.  sol.  of  chloral  hydrate,  and  covered  and 
sealed  and  allowed  to  stand  for  several  days,  when  crystals  of  oxy- 
hemoglobin  appeared.  Note  their  shape  and  color. 

55.  Hemin.     Place  upon  a  slide  a  little  powdered  blood  (from 
a  dried  blood  clot  or  stain),  and  a  few  granules  of  common  salt  (so- 
dium chlorid),  grinding  well  together.     Add  two  or  three  drops  of 
glacial  acetic  acid  and  cover.     Heat  gently  two  or  three  times  until 
the  acid  just  boils,  adding  a  fresh  drop  of  the  acid  after  each  boiling. 
Allow  it  to  cool,  remove  the  cover  and  permit  the  matter  on  the 
slide  and  cover  to  dry.     Examine  both,  dry  and  mount  in  balsam 
the  one  showing  best  the  crystals  of  hemin.     If  there  are  rough 
lumps  of  blood  in  the  preparation,  they  may,  without  injuring  the 
preparation,   be  removed  by   scraping  before  mounting.     A  large 
number  of  hemin  crystals  will  probably  be  found.     Their  shape  and 
color  are  characteristic  and  afford  one  of  the  best  tests  of  blood. 

THE   SPECTRA   OF   BLOOD. 

56.  A  demonstration  of  the  spectra  of  hemoglobin  and  oxy- 
hemoglobin  will  be  given.     Note  the  characteristic  absorption  bands 
of  each  and  their  points  of  occurrence  in  the  spectrum  and  the  dif- 
ferences between  the  spectra  of  hemoglobin  and  oxy-hemoglobin. 
It  is  advisable  to  read  in  some  work  on  physics  about  the  spectro- 
scope and  spectrum  analysis;  also  microscopical  methods,  §§  179- 


TOPICS  FOR  QUIZ— gth  Week. 

THE  BI,OOD  AND  LYMPH  CONTINUED. 

9.  L/eucocytes,  forms  and  amoeboid  movement  ? 

10.  Ingestion  of  foreign  particles  ? 

11.  Fibrin,  (a)  of  blood  ;  (b)  of  lymph  ? 

12.  Blood  crystals,  (a)  hemoglobin  ;  (b)  hemin? 

14.  Medico-legal  importance  of  the  size  of  the  red  corpuscles? 

15.  The  spectrum  of  blood  ? 


37 
THE  MUSCULAR  TISSUES  :  THE  MUSCULAR  SYSTEM. 

LABORATORY    WORK    FOR   THE--       --WEEK. 


References  : 

\ 

Points  for  Quiz  : 

Laboratory  Report  : 

Due: 

Go  the  delivery  desk  for  (a)  assigned  [slide,  69  ;  (b)  a  paraffin 
section,  61  ;  (r)  a  collodion  section,  67  ;  (d)  58;  68,  now  in 
glycerin  ;  mount  as  directed.  (<?)  57  is  in  thin  balsam  ;  mount  a 
drop  of  the  balsam.  (/)  Obtain  63,  64  and  65  and  prepare  them  as 
directed,  (g}  60,  62  and  66  take  in  order. 

NON-STRIATED,  PLAIN   OR   SMOOTH    MUSCLE. 

57.  Muscle    cells    from    the    intestine.      Cat.      Isolated. 
The  muscular  coats  of  the  intestine  were  dissociated  in  nitric  acid 
dissociator  (§  1 1 ) ,  washed  in  water,  the  cells  separated  and  stained 
with  eosin,  now  in   balsam.     Place  a  drop  upon  a  slide  and  apply  a 
cover  glass.     Note  the  length  of  the  muscle  cells,  the  form,  and  size 
of  the  nucleus  in  the  center.     Contraction  nodes  may  also  be  found 
in  the  fibers   (cells)  ;  study   them.     For  the  nucleus  and  striation 
study  59. 

58.  Muscle    cells    from    an    artery.     Cow.     The   uterine 
artery  was  placed  in   nitric  acid  dissociator  (§  n),   the  muscular 
coats  teased  out  to  separate  the  cells.     Stained  in  erythrosin,  now 
in  glycerin  ;  mount  in  glycerin  jelly.     Find  good  examples  of  iso- 
lated cells  and  study,  comparing  them  with  the  cells  in  57  as  to  rela- 
tive length  and  width,  and  the  form  and  size  of  the  nucleus. 

59.  Plain  muscle  cells.     Demonstration.     This  preparation 
is  intended  to  show  especially  (a)  the  shape,  size  and  position  of  the 
nucleus    in  the    cells    of  plain    muscle,    and  (£)  the   longitudinal 
striation  of  the  muscle  fiber.     Compare  57  and  58,  identifying  in 
them  the  nucleus  and,  if  possible,  the  longitudinal  striation. 

60.  Anatomy  of  muscular  coats  (intestine).     Cat.     Obtain 
from  the  delivery  desk  a  small  piece  of  the  intestine,   that  has  been 


38 

macerated  in  nitric  acid  dissociator.  Demonstrate  the  outer  longi- 
tudinal and  inner  circular  coats,  observing  the  relative  thickness  of 
each.  Tease  out  upon  a  slide  the  end  of  a  piece  of  the  longitudinal 
coat,  fraying  the  end  with  a  needle,  and  spreading  it  out  like  a  fan  ; 
cover  in  a  drop  of  water  and  examine.  Observe  the  course  of  the 
fibers  (cells)  and  their  relation  to  each  other.  Compare  with  the 
longisection  and  transection  in  61. 

61.  Transection  and  longisection  of  plain  muscle.     Cat. 
Transection  of  the    intestine.      Picric  alcohol  ;   paraffin  ;    sections 

//.     Stain  with  hematoxylin  15  minutes,  eosin   y?    minute. 
Mount  in  balsam. 

The  outer  muscular  coat  will  afford  a  transection  of  plain  muscle, 
the  inner  coat  a  longisection.  In  the  outer  coat,  note  the  variation 
in  the  size  of  the  sections  of  different  fibers  ;  and  the  occurrence  of 
nuclei  in  only  a  few  ;  why  is  this  ?  Compare  the  section  of  the  inner 
coat  with  the  appearance  found  in  60.  Study  well  the  general 
structural  appearance  of  plain  muscle  in  section  ;  what  are  the  stain- 
ing reactions  of  muscle  and  connective  tissue  with  picrofuchsin  ? 

STRIATED   MUSCLE. 

62.  Fresh  muscle  fibers.     Frog.     Tease  out   upon  a  slide 
in  normal  salt  solution  a  small  shred  of  the  muscle  from  the  leg  of  a 
frog.     Apply  a  cover  glass  and  examine. 

Observe  the  semi-translucent  appearance  of  the  fresh  muscle. 
With  a  y%  objective  study  the  individual  fibers,  noting  the  trans- 
verse striations.  Here  and  there  broken  fibers  may  be  found  ;  ex- 
amine the  broken  ends  to  detect  the  torn  sarcolemma.  Lift  the 
cover  and  add  a  drop  of  2%  acetic  acid,  and  note  the  effect  upon  the 
fibers  in  bringing  out  sharply  the  muscle  nuclei. 

63.  Stained  muscle  fibers.  Macerated    in   acetic 
acid  and  glycerin  and  stained  with  Ehrlich's  hematoxylin  (Sihler's 
method).     Now  in  thin  balsam.     Carefully  separate  the  individual 
fibers  of  the  muscle,  keeping  them  parallel  with  each  other  and 
tearing  them  as  little  as  possible.     Mount  in  balsam. 

The  transverse  striations  and  the  muscle  nuclei  will  show  with 
great  clearness.  Note  also  the  difference  in  caliber  of  different 
fibers. 


39 

64.  Fibrillae.     (Longitudinal  striations).     Biceps  of  rabbit. 
Treated  with  ^%  platinum  bichlorid  6-24  hours  ;  now  in  82%  alco- 
hol.    Carefully  tease   apart  with  needles  the  fibers  of  a  shred  of 
muscle  and  stain   5  minutes  with  erythrosin  ;  dehydrate  and  clear. 
On  the  slide  in  the  clearer  tease  again,  trying  to  split  the  individual 
fibers  ;  you  cannot  tease  too  finely. 

If  the  teasing  is  fine  enough  in  many  places  the  fibers  will  be 
separated  into  their  component  fibrillae.  Observe  this  feature  and 
the  structure  of  the  individual  fibrillae,  the  alternate  light  and  dark 
zones,  lateral  and  transverse  discs,  the  dark  intermediate  disc  in  the 
midst  of  the  light  disc,  and  possibly  the  light  middle  disc  in  the 
midst  of  the  transverse  disc.  Ask  the  instructor  to  show  you  a 
longisection  of  striated  muscle  stained  with  iron  hematoxylin,  in 
which  the  discs  are  more  sharply  differentiated. 

65.  Tendinous  ends  of  muscle   fibers.     Cat.     Intercostal 
muscle,  dissociated  in  nitric  acid  ;  now  in  82%  alcohol.     With  the 
needles  gently  separate  the  fibers  of  a  small  fascicle,  taking  care  to 
have  the  ends  of  the  fibers  well  separated.     If  this  is  successfully 
done,   the  continuity  of  fiber  and  tendon  will  be  shown.     Stain  the 
preparation  with  picrofuchsin  five  minutes,  wash  with  67%  alcohol, 
dehydrate,  clear,  and  mount  in  balsam.     The  muscle  fiber  will  be 
stained  yellow  or  orange,  the  tendon  fibers  red.      Note  the  shape  of 
the  end  of  the  muscle  fiber  and  its  relation  to  the  fibrous  tissue  of 
the  tendon.     Can  you  determine  the  relation  of  the  sarcolemma  at 
the  end  of  the  fiber  ? 

66.  •  Anatomy  of  a  skeletal  muscle.     Obtain  from  the  desk 
an  entire  muscle  which  has  been  macerated  in  20%  nitric  acid.     Dis- 
sect it  carefully,   determining  the  course  and  arrangement  of  the 
component  muscle  fascicles  in  relation  to  the  tendons  (if  present)  of 
origin  and  insertion.     Are  the  fascicles  parallel  with  the  axis  of  the 
muscle  ?     Is  it  a  penniform  or  bipenniform  muscle  ? 

67.  Transaction  of  a  skeletal  muscle.     Cat. 

;  collodion  ;  sections  yu.     Stain  with  hematoxy- 

lin 15  minutes,  picrofuchsin  30  seconds.     Mount  in  balsam.- 

Observe  the  connective  tissue  sheath  of  the  muscle,  the  epi- 
mysium,  the  transected  muscle  fascicles  surrounded  by  the  perimy- 
sium,  the  individual  fibers  held  together  by  the  endomysium  ;  finally, 
in  the  transection  of  the  fibers,  note  the  punctate  appearance  of  the 


40 

cut  ends,  due  to  the  transected  fibrillae,  and  the  muscle  nuclei  entad 
of  the  sarcolemma.  Can  you  recognize  areas  of  Cohnheim  ?  The 
transections  of  the  fibers  are  of  different  sizes.  This  may  or  may 
not  mean  a  difference  in  the  caliber  of  the  fibers.  Why  ?  Compare 
with  61.  You  may  be  shown  transections  stained  with  iron  hema- 
toxylin in  which  the  fibrillae  are  better  differentiated. 

CARDIAC    MUSCLE. 

68.  Muscle    cells.  Isolated    in   caustic 
potash  dissociator  (§  14)  ;  now  in   glycerin.     Place  a  small  drop  of 
the  glycerin  containing  the  cells  upon   the  slide  ;  cover  carefully  ; 
seal  with  shellac. 

Study  the  preparation,  examining  many  cells  and  noting  the 
shape,  the  position  of  the  nucleus  or  nuclei,  the  trans-striations,  the 
relation  of  the  cells  to  each  other,  and  their  processes. 

69.  Transection  of  myocardium.     Human  auricle  or  ven- 
.    tricle.     Assigned   for   study.     Miiller's   fluid  ;    paraffin  ;    sections 

10  /*.     Stained  with  hematoxylin  and  picrofuchsin. 

Observe  the  shape  and  relation  of  the  cells,  the  muscle  columns 
and  fibrillae,  and  the  position  of  the  nucleus  ;  compare  with  the 
transections  of  plain  and  striated  muscle  (61,  67).  You  may  be 
shown  transections  and  longisections  of  cardiac  muscle  stained  with 
iron  hematoxylin,  in  which  the  fibrillae  and  cross-striations  are  bet- 
TOPICS  FOR  QUIZ— loth  Week. 

THE  MUSCULAR  TISSUES  ;  THE  MUSCULAR  SYSTEM. 

1.  Muscular  tissues,  variety  and  characteristics  of  each  ? 

2.  Plain  muscle  cells,  structure?  <^ 

3.  Compare  the  muscle  in  an  artery  and  that  in  the  intestine. 

4.  Name  five  regions  of  distribution  of  plain  muscle. 

5.  Give  relative  length  and  width  of  muscle  cells  in  arteries. 

6.  Compare  plain  muscle  in  transections  and  in  longisections. 

7.  How, distinguish  plain  muscle  from  connective  tissue? 

8.  Stilted  muscle  ;  structure  of  a  muscle  fiber,  considering  sarcolemma, 
muscle  nuclei,  sarcoplasm  and  fibrils? 

9.  Give  the  structure  of  the  fibrillae  and  the  various  discs. 

10.  What  is  the  structure  of  a  skeletal  muscle,  fasciculi,  epi-  peri-  and  endo- 
mysium. 

11.  What  is  the  position  of  muscle  nuclei  in  mammalian  and  immammalian 
muscle  ?     Best  determined  by  transactions. 


THE  NERVOUS  TISSUES  :   THE  PERIPHERAL  NERVOUS 

SYSTEM. 

LABORATORY   WORK    FOR   THE WEEK. 

References  : 
Points  for  Quiz  : 
Laboratory  Report: 

Due: 

Go  to  the  delivery  desk  for  (a)  assigned  slide,  73  ;  (£)  paraffin 
sections,  74,  75,  76,  77,  78  ;  (c}  72,  stained  ;  now  in  clearer;  tease 
as  directed  below  and  mount  in  balsam  ;  (a?)  70  and  71  may  be  pre- 
pared while  the  paraffin  sections  are  being  carried  through. 

NERVE  CELLS. 

70.  From  the  myel  (Spinal  cord).     Calf.     The   myel  was 
split  lengthwise  and  placed  in  formaldehyde  dissociator  (§§  8  and  9). 
Place  a  small  piece  of  the  grey  matter  upon  a  slide  in  a  drop  of  ^% 
eosin  (§  94),  and  proceed  as  directed  in  §  10. 

Search  the  preparation  carefully  until  you  find  a  nerve  cell  with 
quite  long  processes  (dendrites).  Observe  (<z)  the  nucleus  with  its 
nucleolus,  (£)  the  number,  extent,  and  branching  of  the  processes 
of  the  cell-body,  (V)  the  structural  appearance  of  the  cell-body  and 
its  processes.  Can  you  determine  the  identity  of  the  neurite  ?  If 
desired,  you  may  be  shown  nerve-cells  in  section  stained  with, 
methylene  blue,  for  Nissl's  corpuscles. 

71.  From  the  cerebral  cortex.     Cat.     The  hippocamp  was 
cut  up  into  pieces  and  placed  in  formaldehyde  dissociator.     Care- 
fully prepare  two  or  three  preparations  according  to  §  10.     The  best 
preparation  may  be  retained  as  a  permanent  mount. 

Examine  the  slide  carefully,  finding  as  many  cells  with  long 
processes  as  possible.  Compare  with  70,  recognizing  the  identity  of 
structure  of  the  cells,  but  observe  that  these  cells  are  of  a  pyramidal 
form,  with  a  long  apical  process  and  basal  processes.  Can  you  find 
the  neurite  springing  from  the  base  of  the  cell  ? 


42 

NERVE   FIBERS. 

72.  Myelinic  (medullated)  nerve  fibers.      Cat.      Isolated 
fibers  from   the  sciatic  nerve.      Hardened   in    Miiller's  fluid    8-10 
days  ;  hardened  in  alcohols  ;  dehydrated  ;  in  chloroform  1-2  days  ; 
95%  alcohol  ;  82%  alcohol  ;  water  ;  stained  in  Delafield's  hematoxy- 
lin   12-24  hours  (§  82)  ;  now  in  clearer.      With  needles  tease  out  a 
small  bundle  of  fibers,  keeping  them  parallel  if  possible.      Mount  in 
balsam. 

In  studying  this  preparation,  determine  the  axis  cylinder,  the 
myelinic  sheath,  the  nodes  of  Ranvier,  the  internodes, — their 
length, — the  nerve  corpuscles  (nuclei).  Can  you  recognize  the 
neurilemma  ?  Does  the  preparation  show  medullary  segments  or  a 
reticular  structure  of  the  myelinic  sheath  ? 

73.  Amyelinic  nerve  fibers.     Ox.     Isolated  fibers  from  the 
splenic  nerve.     Assigned  for  study.     Dissociated  in  -£$%  osmic  acid 
(§  17)  ;  teased  apart  with  needles. 

Both  myelinic  and  amyelinic  fibers  are  present,  and  care  must 
be  exercised  in  distinguishing  between  delicate  myelinic  fibers  and 
the  amyelinic  ones.  When  a  good  place  is  found,  study  the  amye- 
linic fibers,  noting,  as  compared  with  the  myelinic  nerve-fiber,  the 
absence  of  the  myelinic  sheath  and  the  greater  number  of  nerve 
nuclei.  Compare  the  myelinic  fibers  in  this  preparation  with  those 
in  72.  The  myelin  of  the  sheath  is  retained  and  blackened  in  73  ; 
in  72  it  has  been  dissolved  out. 

PERIPHERAL   NERVES. 

74.  Sciatic      nerve.       Cat.       Transection  ;       chrome-oxalic 
(§  32)  >    paraffin  ;    sections  //.     Stain   with  hematoxylin    15 
minutes,  picrofuchsin  ^2  minute.     Mount  in  balsam. 

Study  this  preparation  for  (a)  the  structure  of  a  nerve  trunk, 
noting  its  component  funiculi  or  bundles,  surrounded  by  connective 
tissue  sheaths,  the  perineurium,  and  bound  together  by  the  epi- 
neurium,  while  within  the  funiculi,  the  individual  nerve  fibers  with 
the  endoneurium  ;  (£)  the  structure  of  myelinic  nerve  fibers  in  tran- 
section,  showing  the  central  axis-cylinder,  surrounded  by  the  mye- 
linic sheath  (the  myelin  dissolved  out).  Find  a  place  where  a 
nerve  nucleus  is  cut.  Compare  with  the  transection  of  a  muscle, 
recognizing  the  analogy  in  the  relations  of  the  connective  tissue. 


43 

75.  Ulnar     nerve.  .       Transaction.       Hermann's 
fluid  (§23)  ;  paraffin  ;  sections  yw.     Stain  for  2-3  minutes  with 
picrofuchsin. 

This  will  be  a  supplementary  preparation  to  74,  since  here 
the  myelin  is  not  dissolved  out  and  is  stained  black  by  the  osmic 
acid.  Compare  the  two  preparations,  recognizing  in  this  one  the 
features  enumerated  above. 

76.  Vagus    and     sympathic.  Transection  ;     Her- 
mann's  fluid    (§  23)  ;    paraffin.       Stain    2-3    minutes  with  picro- 
fuchsin. 

There  will  also  be  included  a  transection  of  the  carotid  artery. 
Note  the  myelinic  nerve  fibers,  with  blackened  myelinic  sheaths  in 
both  nerves  and  in  the  vagus,  the  variation  in  their  caliber.  In  ad- 
dition, recognize  if  possible  the  transected  amyelinic  fibers  by  the 
absence  of  a  myelinic  sheath. 

GANGLIA. 

77.  Sympathetic  ganglion.      Cat.     Section  of  the  semilunar 
ganglion.     Zenker's   fluid  ;    paraffin  ;    sections  /u.     Stain    i 
hour  with  hematoxylin,  picrofuchsin  30  seconds.     Observe  well  the 
characteristic  appearance  of  ganglion  cells  in  section  ;  their  finer 
structure,  indications  of  their  processes  ;  recall  the  structure  of  the 
nerve  cells  in  70  and  71,  realizing  the  extent  of  the  dendrites. 

78.  Spinal  ganglion.  Section  of  the  ganglion  upon 
the  dorsal   root  of  a  spinal  nerve.      Flemming's  fluid  ;    paraffin  ; 
sections             yu.     Stain  with  hematoxylin  i   hour,  picrofuchsin  30 
seconds.     Observe  the  characteristic  appearance  of  a  ganglion,  the 
(apparently)  round  ganglion  cells  with  their  capsules  ;  the  nerve 
fibers  ;  the  connective  tissue  within  the  ganglion  and   forming  a 
covering  sheath.     Compare  with  77.     What  is  the  real  form  of  the 
nerve  cells  in  the  two  kinds  of  ganglia  and  their  relation  to  the 
fibers  ? 

PERIPHERAL   NERVE    ENDINGS. 

79.  Motor  end-plate.  From  the  muscle. 
Demonstration.     The  preparation  will  show  the  nerve  fiber  termin- 
ating in   the   end-plate   with    its   nuclei   and   the   branching  axis- 
cylinder. 


44 

80.  Sensory.     Free  nerve  endings  in 
Demonstration.     Stained  by  Golgi's  rapid  method. 

Illustrates  the  branching  and  free  termination  of  the  axis-cylin- 
der in  an  epithelium. 

81.  Pacinian     corpuscles.       Cat.        From    the    mesentery. 
Demonstration.     Note  the  core  (inner  bulb)  in  which  the  axis-cyl- 
inder of  thenerve  fiber  terminates   and  the  concentric   connective 
tissue  lamellae  of  the  capsules. 

82.  Meissner's  corpuscles.     From  the  human  finger.     Dem- 
onstration.    There  is  shown  a  corpuscle  in  one  of  the  papillae  of 
the  skin.     Observe  the  form  and  structural  appearance. 

Examine  17  for  transected  corpuscles  in  the  papillae  of  the  skin. 
Personal  preparations  showing  81  and  82  will  probably  be  obtained 
later.  (149,  153). 


TOPICS  FOR  QUIZ— nth  Week. 

THE  NERVOUS  TISSUES  ;  THE  PERIPHERAL  NERVOUS  SYSTEM. 

1.  Distinction  (a)  central,  (b)  peripheral  nervous  systems. 

2.  What  are  neurones,  nerve  cells,  nerve  fibers,  neuroglia? 

3.  What  is  the  relation  of  nerve  fiber  and  nerve  cell  ? 

4.  Structure  of  a  nerve  cell  considering  :  (a)  Nucleus,  (b)  cell-body,  (c) 
processes,  neurites  and  dendrites. 

5.  What  is  the  structure  of  the  nerve  cells  in  (a)  a  spinal  ganglion,  (b)  a 
sympathetic  ganglion? 

6.  Give  two  general  types  of  nerve  cells. 

7.  Nerve  fibers,  varieties. 

8.  Structure  of  a  myelinic  nerve  fiber,  axis   cylinder,    myelinic   sheath, 
neurilemma,  nerve  nuclei  or  corpuscles? 

9.  What  are  nodes  of  Ranvier  ?    Internodes  ? 

10.     Amyelinic  nerve  fibers  ;  structure  compared  with  myelinic  nerve  fibers. 

n.     Where  found,  at  least  two  places? 

12.     How  is  the  nerve  impulse  transmitted  from  one  nerve  cell  to  another? 


45 
THE  BLOOD  AND  LYMPH  VASCULAR  SYSTEM. 

LABORATORY    WORK    FOR   THE  -1^1    -WEEK.       %^ 


References  :_  .  .  .  V(l(  ^         -  _vv< 


Points  for  Quiz  : 
Laboratory  Report  : 

Due: 

v^ 

Obtain  from  the  delivery  desk  (a)  assigned  slide,  85  ;  (£) 
paraffin  sections,  83,  84,  86,  92,  94  ;  (c)  collodion  sections,  91,  95  ; 
(*0  93>  already  stained. 

ARTERIES. 

83.  Muscular  artery.      Cow.      Transection   of  the   uterine 
artery.     Zenker's  fluid  (§  28)  ;  paraffin  ;  sections    '  /*.     Stain 
with  hematoxylin  30  minutes,  picrofuchsin  30  seconds. 

In  this  preparation  identify  the  three  coats  forming  the  walls  of 
arteries  and  veins, — the  intima,  media  and  adventitia  ;  noting  the 
relative  thickness  of  each.  In  the  intima,  recognize  the  vascular 
epithelium,  the  internal  elastic  membrane,  and  the  subepithelial 
connective  tissue.  In  the  media  should  be  found,  plain  muscle  cells, 
elastic  bands  and  white  connective  tissue  ;  recall  the  staining  reac- 
tions of  picrofuchsin  (used  for  this  preparation)  with  muscle,  white 
and  elastic  connective  tissue.  Are  there  longitudinal  muscular 
bundles  present  ?  Note  the  relative  amounts  of  white  and  elastic 
tissue.  In  the  adventitia,  observe  the  relative  amounts  and  arrange- 
ment of  the  white  and  yellow  elastic  fibers.  Is  an  external  elastic 
membrane  present  ? 

84.  Elastic  artery.  Transection  of  the  carotid 
artery.    CVJ,                          ;  paraffin  ;  sections        \       p.     Stain  with 
hematoxylin             minutes,  picrofuchsin  seconds. 

Compare  this  preparation  with  83,  recognizing  the  coats,  their 
relative  thickness  and  structure.  Especially  compare  the  media  in 
the  two  preparations,  noting  the  relative  amounts  of  muscle  and 
elastic  tissue  in  each. 


46 

85.  Aorta.     Homo.     Transection  or  longisection  of  the  aorta. 
Assigned  for  study.     Picric  alcohol ;  paraffin  ;  stained  with 
hematoxylin  and  picrofuchsin. 

Study  carefully  in  comparison  with  83  and  84.  Are  internal  or 
external  elastic  membranes  present  ?  What  is  the  relative  thickness 
of  the  coats  and  the  relative  amounts  of  muscle  and  elastic  tissue  in 
the  media  ?  Are  vasa  vasorum  to  be  seen  in  the  adventitia  ? 

VEINS. 

86.  Femoral  vein  (and  artery).  Transection  of  the 
femoral  artery  and  vein.                           ;  paraffin  ;  sections  fj. . 
Stain  with  hematoxylin             minutes,  picrofuchsin               seconds. 

Identify  the  artery  from  your  knowledge  of  its  structure  as 
gained  from  a  study  of  83,  and  compare  with  it  the  vein,  observing 
the  differences  and  resemblances  in  the  following  particulars  :  (a) 
the  thickness  of  the  wall,  (£)  its  flaccidity,  (c)  the  presence  of  blood 
in  the  lumen,  (d}  relative  thickness  of  the  coats,  (e)  relative 
amounts  of  white  connective  tissue,  elastic  tissue  and  muscle  pres- 
ent. From  a  study  of  their  structure,  compare  arteries  and  veins  as 
to  their  elasticity  and  contractility. 

CAPILLARIES. 

87.  Vascular    epithelium.  Demonstration    of    the 
vascular  epithelium  as  seen   in  capillaries  and  arterioles.     Silvered 

(§  146). 

Note  the  shape  of  the  cells  as  outlined  by  the  blackened  cell- 
cement  ;  their  relative  size  in  the  capillaries.  Remember  that  the 
vascular  epithelium  is  the  structure  common  to  all  parts  of  the  vas- 
cular system. 

88.  Capillaries  in  striated  muscle.     Demonstration.     Free- 
hand longitudinal  section  of  muscle  that  was  injected  with  carmine 
gelatin  mass  (§  127). 

Study  carefully  the  capillary  network  and  its  relation  to  the 
muscle  fibers. 

89.  Capillaries  of  plain  muscle.     Rabbit.     Demonstration. 
The  blood  vessels  of  the  small  intestine  were  injected  with  carmine 
gelatin  mass  (§  127),  the  muscular  coats  stripped  off,  pieces  of  the 
longitudinal  coat  laid  out  flat.     Balsam  mount. 


47 

Note  the  distribution  of  the  vessels  in  the  muscular  tissue,  the 
shape  of  the  capillary  network,  and  compare  it  with  88. 

90.  Capillaries    of    an    organ.  Demonstration. 
Blood    vessels    injected    with   carmine   gelatin   mass ;  hardened  in 
alcohol  ;  collodion. 

Note  the  capillary  network  and  the  great  vascularity.  The 
vascularity  of  the  different  organs  will  be  considered  subsequently 
with  the  organs  themselves. 

LYMPHATIC  TISSUES. 

91.  Peyer's  patch.     Cat.     Transection  of  the  ileum.     vom 
Rath's  fluid  ;  collodion  ;  sections    10     JJL.     Stain  with  hematoxylin 
15  minutes,  picrofuchsin  15  seconds. 

The  section  passes  through  a  Peyer's  patch.  In  the  villi  and 
mucosa  will  be  found  diffuse  adenoid  (lymphatic)  tissue.  Peyer's 
patch  is  an  aggregation  of  lymphatic  nodules,  "dense"  lymphatic 
tissue.  Note  carefully  the  general  structural  appearance  of  adenoid 
tissue,  the  character  of  the  lymph  cells  and  their  nuclei.  Can  you 
recognize  the  lymphoid  reticulum  ?  Does  the  preparation  show  the 
relation  of  the  lymphatic  tissue  to  the  epithelium  ? 

92.  Tonsil.     Dog.     Section  of  the  tonsil.     Zenker's   fluid  ; 
paraffin  ;  sections  JJL.     Stain  the  section  with  hematoxylin  30 
minutes,  picrofuchsin  30  seconds. 

Study  the  preparation,  recognizing  that  it  is  composed  of  ade- 
noid tissue,  consisting  of  nodules  surrounded  by  diffuse  adenoid 
tissue.  Identify  its  structure  with  that  of  Peyer's  patch.  Note  the 
relation  of  the  lymphatic  tissue  to  the  overlying  stratified  epithelium 
of  the  oral  cavity  ;  is  the  epithelium  destroyed  or  filled  with  lymph 
cells  ;  are  there  any  lymph  cells  upon  the  surface  of  the  epithelium  ? 

93.  Lymphatic  gland.    Cat.    Transection.    vom  Rath's  fluid  ; 
stained  in  toto  in  paracarmine  ;  collodion  ;  sections  yu.     Mount 
directly. 

Study  the  general  structural  appearance  of  the  gland.  Identify 
the  following  regions  or  parts  :  (a)  the  cortex,  medulla  and  the 
hilum  ;  (£)  the  lymphatic  nodules  (follicles)  in  the  cortex  ;  (c)  the 
capsule  and  the  trabeculae  ;  can  you  find  plain  muscle  in  the  cor- 
tex ?  (d)  blood  vessels.  Compare  the  lymphatic  gland  with  91 
and  92.  Can  you  recognize  the  lymph  sinuses  bordering  the  trabe- 


48 

culae  ?     Understand  the  relation  of  the  afferent  and  efferent  lymph 
vessels  to  the  gland  and  the  course  of  the  lymph  through  it. 

94.  Thymus.  Transection.  ;  paraffin  ;  sec- 
tions     •       IJL.     Stain  with  hematoxylin  15  minutes,  picrofuchsin  30 
seconds. 

Compare  this  preparation  carefully  with  93,  identifying  as  be- 
fore, capsule,  trabeculae,  cortex  and  medulla,  lymph  follicles  (nod- 
ules), and  in  addition,  the  lobular  structure.  Can  you  find  corpus- 
cles of  Hassall  ;  what  is  their  significance?  Study  the  structural 
appearance  of  the  organ  so  that  you  may  distinguish  it  from  93  and 
95.  Study  the  microscopic  appearance  of  the  thymus. 

95.  Spleen.      Dog.      Transection.      vom  Rath's  ;  collodion  ; 
sections  yw.     Stain  with  hematoxylin  15  minutes,  picrofuchsin 
30  seconds. 

Examine  the  gross  preparations  of  the  spleen,  observing  care- 
fully the  structural  appearance  of  natural  and  cut  surfaces.  Study 
the  microscopic  preparation,  recognizing  (a)  the  capsule  and  the 
trabeculae  ;  is  plain  muscle  present  in  these  ?  (£)  the  splenic  pulp 
containing  the  Malpighian  corpuscles  (lymphatic  follicles  or  nod- 
ules), and  blood  vessels.  Examine  the  Malpighian  corpuscles,  not- 
ing their  structure  and  the  eccentric  artery  ;  compare  them  with  the 
lymphatic  nodules  in  93  and  94.  '  For  the  supporting  framework  of 
the  spleen,  the  capsule  and  trabeculae,  examine  the  gross,  mace- 
rated preparation.  Study  carefully  the  general  structural  appear- 
ance of  the  section  of  spleen,  so  that  you  may  distinguish  it  from 
lymph  gland  or  thymus.  Again  consult  the  gross  section  of  spleen, 
identifying  capsule  and  trabeculae,  Malpighian  corpuscles  and 
splenic  pulp.  Understand  the  relation  of  the  blood  vessels  to  the 
Malpighian  corpuscles  and  to  the  pulp. 


49 
THE  DIGESTIVE  SYSTEM. 

LABORATORY    WORK    FOR    THE-^J? WEEK.          ''"*   '  ^d 

References:   L^  •  *  -^  - 
Points  for  Quiz  : 
Laboratory  Report :     \IVx>.-\ 


Go  to  the  delivery  desk  for  (a)  assigned  slide  100,  (£)  paraffin 
sections  103,  108,  (V)  collodion  sections  96,  97,  101,  104,  (d) 
98,  99,  102,  105,  1 06,  already  stained  and  in  clearer. 

THE   ORAL   CAVITY. 

(10).  Epithelium,  glands.  Section  of  the  soft  palate,  cat. 
Assigned  for  study  (10).  The  oral  side  of  the  soft  palate  affords  a 
demonstration  of  the  stratified  epithelium  of  the  oral  cavity  ;  study 
its  appearance.  In  the  substance  of  the  soft  palate  are  numerous 
mucous  glands,  which  open  upon  the  mucosa  of  the  oral  cavity. 
Serous  glands  are  not  present.  Study  their  appearance  and  struct- 
ure ;  find  if  possible  a  duct  leading  to  the  surface.  These  would 
belong  to  the  group  of  palatine  glands  ;  others  would  be  lingual, 
buccal  or  labial,  according  to  their  location. 

96.  Developing  tooth.  Early  stage.  Transection 

through  the  cephalic  part  of  the  head  of  an  embryo  long. 

;  collodion  ;  sections  yw.  Stain  with  hematoxylin 

and  eosin. 

Within  the  section  will  appear  the  two  nasal  cavities  and  below 
them  the  oral  cavity,  with  the  tongue  projecting  up  from  the  floor 
of  the  mouth.  On  each  side,  opposite  the  developing  lower  and 
upper  jaws,  will  be  seen  the  dental  ridge  projecting  from  the  epi- 
thelium of  the  oral  cavity.  One  of  the  four  sections  of  dental  ridge 
will  probably  pass  through  a  developing  tooth,  showing  the  cap- 
like  enamel  organ  covering  the  papilla  which  forms  the  dentine  and 
pulp  of  the  adult  tooth. 


50 

97.  Developing    tooth  ;     later    stage.      Dog.      Transection 
through  the  lower  jaw  of  a  new  born  puppy.     Picric  alcohol  ;  decal- 
cified (§  131)  ;  collodion;  sections    v      /*.     Stain  with  hematoxy- 
lin  and  picrofuchsin. 

Be  sure  your  section  passes  through  a  developing  tooth.  In 
this  more  mature  tooth  there  should  be  recognized  (a)  the  enamel 
organ  with  its  three  layers,  (£)  the  enamel,  (c}  the  dentine  lined 
by  the  odontoblasts,  and  finally  (d),  the  dental  pulp,  containing 
blood  vessels. 

98.  Tongue.    Rabbit.     Transection.     Miiller's  fluid  ;  stained 
in  toto  (§  74  c)  ;  collodion  ;  sections     .  u     J*- 

Study  the  preparation,  noting  the  surface  epithelium  with  low 
papillae,  in  the  body  of  the  tongue,  the  mesal  septum  and  the  bun- 
dles of  muscle  in  general  running  vertically,  longitudinally  and 
transversely  ;  also  nerves  and  blood  vessels. 

ESOPHAGUS. 

99.  Esophagus.     Dog.     Transection.   c 
sections  jj..     Stained  in  toto.     ^P  &r< 

In  this  preparation,  as  well  as  in  the  succeeding  regions  of  the 
digestive  tract,  identify  the  four  coats — mucosa,  submucosa,  (inner 
and  outer)  muscular  and  serous  coats.  In  the  mucosa  and  sub- 
mucosa observe  the  character  of  the  lining  epithelium,  the  mucous 
glands  ;  find  if  possible  a  duct  opening  upon  the  lining  epithelium  ; 
the  muscularis  mucosa  ;  the  character  and  direction  of  its  fibers.  In 
the  muscular  coats  determine  the  character  and  direction  of  the 
muscle  fibers.  What  differences  are  there  in  different  animals  in 
the  muscular  coats  of  the  esophagus  ? 

100.  Esophagus.     Homo   or    sheep.     Assigned    for    study. 
Transection. 

Compare  with  99,  recognizing  the  coats,  the  epithelium,  mus- 
cularis mucosa,  character  and  relation  of  the  muscular  fibers  in  the 
muscular  coat.  Are  glands  present  ? 

STOMACH. 

101.  Stomach.     Dog.     Vertical  section   of  the  wall,  cardiac 
end  near  the  esophagus.  X  o>rv  NfL^vv     >  collodion;  section     '.\    /*. 
Stain  the  sections  with  hematoxylin  and  eosin. 


Identify  as  before  the  four  coats,  noting  the  character  of  the 
muscular  tissue  in  the  muscular  coat,  the  number  of  layers  recog- 
nizable in  it  and  in  the  muscularis  mucosae.  In  the  submucosa 
observe  the  presence  of  blood  vessels,  both  arteries  and  veins.  In 
the  mucosa  are  the  tubular  gastric  glands  opening,  two  or  three  to- 
gether, into  depressions  of  the  surface  epithelium.  In  these  gland 
tubules  recognize  the  two  kinds  of  cells,  chief  and  border  cells  ;  re- 
call the  function  of  each.  Compare  the  surface  epithelium  of  the 
stomach  with  that  of  the  esophagus.  If  your  preparation  does  not 
show  well  the  gland  tubules  and  their  cells  ask  the  instructor  to 
show  you  one  that  does. 

102.  Stomach.     Dog.      Vertical   section    through    the   wall, 
pyloric  end.  ^U^J^SL^  ',  stained  in  toto  in  paracarmine  ;  collodion  ; 
sections      .  f     j*. 

Compare  this  preparation  with  101,  recognizing  the  coats  and 
their  structure  and  noting  differences  in  the  muscular  coats,  espe- 
cially the  increase  in  the  thickness  of  the  inner  circular  coat,  and  in 
the  glands  of  the  mucosa.  Observe  that  the  pyloric  glands  differ 
from  the  peptic  (oxyntic)  glands  shown  in  101  in  (a)  the  absence  of 
parietal  cells,  (£)  the  longer  duct  into  which  several  tubules  open. 
Recall  the  relative  extent  of  the  regions  occupied  by  the  two  kinds 
of  glands. 

103.  Zymogen    granules.     Calf.     Vertical    section    through 
the   mucosa  of  the  cephalic  end  of  the  stomach  (fourth  stomach  or 
abomasum).       i%    osmic    acid;     paraffin;     sections    'If      ft.     No 
staining  or  Hcl.  carmine  (§  87). 

In  the  deeper  part  of  the  mucosa  will  be  seen  the  gastric 
tubules,  cut  across  in  various  directions  and  lengthwise.  The  ends 
of  the  cells  forming  the  tubules — the  chief  cells —  toward  the  lumen 
of  the  tubule  will  be  seen  crowded  with  granules  of  a  yellowish 
brown  color.  These  granules  are  the  precursors  of  the  ferment,  or 
pepsinogen  granules.  Find  a  border  cell  and  note  that  no  such 
granules  are  contained  in  it. 

SMALL   INTESTINE. 

104.  Duodenum.     Cat.     Transection.     Zenker's  fluid  ;  col- 
lodion ;    sections     ' ,  ^     yu.     Stain    with    hematoxylin    and    picro- 
fuchsin. 


52 

In  studying  this  preparation,  observe  that  the  coats  recogniz- 
able in  the  esophagus  and  stomach,  also  form  the  intestine.  Deter- 
mine the  character  and  direction  of  the  fibers  in  the  two  layers  of  the 
muscular  coat.  Is  a  muscularis  mucosa  recognizable  ?  In  the  mu- 
cosa,  note  the  tubular  crypts  of  Lieberkiihn  extending  down  into  the 
mucosa,  and  the  villi,  tongue-like  elevations  of  the  mucosa.  Study 
the  character  of  the  epithelium  of  the  intestine,  noting  the  striated 
border  of  the  columnar  cells  and  the  goblet  cells.  Compare  the  epithe- 
lium covering  the  villi  with  that  of  the  crypts  of  Lieberkiihn.  In 
the  submucosa  are  the  glands  of  Brunner  with  here  and  there  a  duct 
leading  through  the  mucosa.  What  relation  do  they  have  to  the 
pyloric  glands  ?  Study  carefully  the  structure  of  a  villus  ;  (a)  its 
covering  epithelium,  (£)  the  central  core  of  diffuse  adenoid  tissue, 
(r)  a  strand  of  plain  muscle  ;  and  in  comparison,  study  105,  106, 
and  6. 

105.  Duodenum.     Rabbit.     Injected  with    carmine    gelatin 
mass  (§  127)  ;    now  in  clearer.     Mount   a   piece    of  the   mucosa, 
villi  up. 

The  villi  are  rather  short  and  leaf-like,  flattened.  Observe  how 
the  arteriole  passes  up  to  near  the  summit  of  the  villus,  there  to 
break  up  into  a  cascade  of  capillaries  that  unite  on  the  opposite  side 
to  form  one  or  two  venules. 

1 06.  Ileum.     Rabbit.     Transection.     Injected  with  carmine 
in  mass  (§  127);    alcohols;  collodion;    now   in  clearer;  sec- 
tions   \vr\>    yu. 

In  the  ileum  the  villi  are  longer  and  more  filiform.  Note  the 
relations  of  the  blood  vessels  in  the  villi  and  compare  with  105. 

107.  Ileum.      Rabbit.      Demonstration.     Blood    vessels    in- 
jected with  carmine  gelatin  mass  (red)  ;  the  central  lacteal  is  filled 
with  Berlin  blue  gelatin  mass  (blue).     Observe  the  position  of  the 

-  lacteal  (lymph  vessel)  in  the  villus,  its  extent  and  size  in  compari- 
son with  the  blood  capillaries.  By  means  of  6,  104,  106  and 
107  a  fairly  complete  idea  may  be  formed  of  the  structure  of  a  villus  ; 
from  your  physiology  recall  the  part  played  by  the  epithelium  in 
absorption  and  what  food  stuffs  pass  into  the  lacteal  and  what  are 
taken  up  by  the  blood  vessels. 

1 08.  Fat    absorption.     Intestine  of  the  frog.     Flemming's 
0  >j        fluid  (§  24)  ;  paraffin.     The  frog  was  fed   with  bacon  24  hours  be- 


5.3 

fore  it  was  killed,  and  the  fat  is  being  absorbed  by  the  epithelial 
cells  ;  the  globules  in  the  epithelium  are  blackened  by  the  osmic 
acid  of  the  Flemming's  fluid.  If  desired  the  preparation  may  be 
stained  with  safranin  (§§  89,  99). 

The  intestine  of  the  frog  has  no  villi,  the  elevations  of  the 
mucosa  shown  in  the  transection  are  folds  cut  across.  The  epi- 
thelial cells  covering  the  villi  in  mammals,  however,  have  the  same 
function  in  the  absorption  of  fat  (6). 

(91).  Ileum.  Cat.  Re-examine  this  preparation  in  the  light 
of  the  knowledge  gained  of  the  structure  of  the  intestine,  noting 
the  lymph  follicles  and  their  relation  to  the  intestinal  epithelium  ; 
the  occurrence  of  lymph  cells  in  and  upon  the  epithelium.  Identify 
also  the  coats  and  structures  mentioned  under  104,  save,  of  course, 
Brunner's  glands,  which  are  limited  to  the  cephalic  part  of  the 
duodenum. 


TOPICS    FOR    QUIZ— isth    Week. 

THE  DIGESTIVE  SYSTEM. 

1.  Constituents  of  the  digestive  system? 

2.  Digestive  organs  in  or  communicating  with  the  oral  cavity  ? 

3.  Structure  of  the  wall  of  the  oral  cavity  ? 

4.  Structure  of  the  tongue  ? 

5.  Structure  of  a  tooth  ?     Development  of  a  tooth. 

6.  Structure  of  the  .esophagus  (a)  different  regions  in  man,  (b)  in  the 
domestic  animals. 

7.  Cardiac  stomach.     Pyloric  stomach.     Stomach  of  man,  horse,  rat,  pig, 
ox  and  sheep. 

8.  Small  intestine.     Glands   there   found?     How  distinguish   trans-and 
longi-sections  ? 

9.  Injected  small  intestine  ;  lymphatics  and  blood  vessels. 
10.     Fat  absorption  and  zymogen. 


54 
THE  DIGESTIVE  SYSTEM. 

LABORATORY   WORK    FOR   THE--'-^ WEEK.       1 1.  -  '<-.*•) 

References  :    C\\ .  *     ^-   i  v«»<*  -  i  «o ^ 
Points  for  Quiz : 

Laboratory  Report  : 

Due:    'JOb.  6^ 

Go  to  the  delivery  desk  for  (a)  assigned  slides,  no,  114,  118  ; 
(b)  paraffin  sections,  112,  113,  115  ;  (c)  collodion  sections,  109,  116, 
117  ;  (d)  sections  already  stained,  in. 

LARGE   INTESTINE. 

109.  Colon.  (L^A-  Transection.  Picric  alcohol  ;  collodion  ; 
sections  .  >  yw.  Stain  with  hematoxylin  and  picrofuchsin. 

Compare  with  the  sections  of  small  intestine,  noting  identity  of 
structure  of  the  coats  and  their  general  structure  and  relations,  dif- 
ferences in  the  absence  of  villi,  greater  number  of  ccrypts  of  Lieber- 
kiihn,  increase  in  the  relative  number  of  goblet  cells. 

no.  Rectum.  Kitten.  Transection.  Assigned  for  study. 
;  collodion  ;  sections  *  (  .ju.  Stained  with 

Compare  this  preparation  with  109,  observing  especially  the 
increased  thickness  of  the  muscular  coats  and  the  muscularis  mu- 
cosae,  and  the  looser  connection  of  the  mucosa  with  the  muscular 
coat. 

in.  Caecum.  Rabbit.  Blood  vessels  injected  with  carmine 
gelatin  mass  ;  now  in  clearer  ;  mount  in  balsam,  rough  side  (mu- 
cosa) up.  Note  the  capillary  net  work  and  its  denser  arrangement 
in  small  areas, — rudimentary  villi. 

SALIVARY   GLANDS. 

112.  Mucous  type.  Submaxillary  gland.  Cat.  Mercuric 
chlorid  -f-  5%  glacial  acetic  acid  (§  26);  paraffin  ;  section  li  /u. 
Stain  with  hematoxylin  and  picrofuchsin. 

Study  the  section  carefully,  noting  (a)  the  general  structural 
appearance  of  the  gland,  its  lobulation,  (£)  the  component  acini  and 


55 

ducts,    (V)  the  blood  vessels  and  nerves  (and  ganglion),  together 
with  the  connective  tissue. 

Study  carefully  the  character  of  the  cells  composing  the  secret- 
ing acini,  noting  (a)  the  structure  and  staining  reaction  of  the  cell- 
body,  (b)  the  position  and  structure  of  the  nucleus.  Can  you  find 
a  place  where  duct  and  secreting  portion  are  continuous  ?  Recog- 
nize the  presence  of  demilunes  and  note  the  character  of  the  cells. 

113.  Serous  type.     Section  of  the  parotid  gland.    Cat.    Mer- 
curic  chlorid  +  5%   glacial   acetic  acid   (§  26)  ;  paraffin  ;  sections 

1^     //.     Stain  with  hematoxylin  and  picrofuchsin. 

Study  the  section  for  the  general  structural  appearance  and 
lobulation,  the  secreting  acini  and  ducts,  blood  vessels,  nerves  and 
connective  tissue.  Study  the  acini  carefully,  comparing  them  with 
the  secreting  acini  in  1 1 2  as  to  the  shape  and  size  of  the  cells,  the 
position  and  structure  of  the  nucleus,  and  the  structure  and  appear- 
ance of  the  cell  bodies.  What  are  the  differences  ?  Can  you  dis- 
tinguish mucous  and  serous  glands  ?  Compare  the  glands  you 
found  in  the  esophagus  and  soft  palate. 

PANCREAS. 

114.  Pancreas.     Horse.     Assigned  for  study.     Picro-forma- 
lin  ;  paraffin  ;  hematoxylin  and  eosin. 

Examine  the  gross  preparations  for  general  appearance,  lobula- 
tion, compactness,  color,  etc. 

In  studying  the  section  analyze  its  structure  in  the  same  man- 
ner as  112  and  113,  observing  carefully  its  general  structural  ap- 
pearance, the  ducts  and  secreting  acini  (tubules),  the  structure  and 
shape  of  the  cells  forming  these,  the  position  of  the  nucleus.  Are 
areas  of  L,angerhans  present  in  the  section  ?  Compare  the  section 
with  113  as  to  (a)  compactness,  ($)  shape  of  the  secreting  acini, 
(<:)  the  lumen  of  the  acini.  Can  you  distinguish  them  ? 

115.  Zymogen  granules.     Pancreas.     Calf.      i%  osmic  acid 
(§22);  paraffin;  sections       I.     /^.     No  staining. 

In  the  ends  of  the  cells  forming  the  secreting  acini  (tubules) 
observe  the  presence  of  light  brown  granules —  trypsinogen  gran- 
ules, precursor  of  the  ferment  secreted  by  the  gland.  Compare  103. 

116.  Injected    pancreas.     Rabbit.      Blood   vessels   injected 
with  carmine  gelatin  mass  ;  alcohols  ;  collodion  ;  stain  lightly  with 
hematoxylin.     This  preparation  illustrates  the  vascularity  of  the 


56 

gland  and  the  relation  of  the  capillary   network   to  the  tubules  of 
the  acini  of  the  gland. 

LIVER. 

117.  Liver.     Pig.     Picric  alcohol  ;  collodion;  sections         yu. 
Stain  with  hematoxylin  and  picrofuchsin. 

Study  thoroughly  the  preparations  illustrating  the  gross  anat- 
omy of  the  liver  ;  («)  position,  (£)  aspects,  (V)  lobes,  (d)  the  longi- 
tudinal and  transverse  fissures,  (>)  the  appearance  of  the  liver  sub- 
stance as  seen  in  section  and  the  natural  surface  (covered  by  the  con- 
nective tissue  capsule),  (/)  the  indistinct  lobulation. 

In  studying  the  section  of  liver  note  the  lobules  separated  by 
connective  tissue  septa.  Within  the  lobules  recognize  the  intra- 
lobular  vein  and  the  arrangement  of  the  liver  cells.  In  the  inter- 
lobular  connective  tissue  will  be  found  inter-lobular  veins,  inter- 
lobular  arteries,  small  bile  ducts.  Can  you  find  intra-lobular  blood 
capillaries  passing  off  from  the  inter-lobular  veins  or  entering  the 
intra-lobular  veins  ?  Be  sure  you  understand  the  course  of  the 
blood  and  the  origin  and  relations  of  the  different  vessels.  What  is 
the  relation  of  the  liver  cells  to  the  blood  capillaries  and  the  bile 
capillaries  ?  Note  well  the  structural  appearance  of  the  liver  so 
that  you  can  distinguish  it  from  other  glands.  The  liver  of  the 
pig  has  a  large  amount  of  connective  tissue  between  the  lobules,  sep- 
arating them  completely  ;  hence  their  distinctness. 

118.  Liver.     Horse  or  Homo.     Assigned    for  study.     Potas- 
sium dichromate  or  Krlicki's  fluid  ;  paraffin  ;  stained  with  hema- 
toxylin and  picrofuchsin. 

Study  carefully,  identifying  the  parts  and  structures  already 
recognized  in  117;  compare  with  that  preparation,  noting  the  in- 
complete isolation  of  the  lobules,  less  inter-lobular  connective  tissue, 
etc.  Again  consult  the  gross  preparations,  examining  them  in  the 
light  of  your  present  knowledge  of  the  structure  of  the  liver. 

119.  Bile  capillaries.     Liver  of  ox.     Demonstration.     The 
bile  capillaries  are  gorged  with  bile,  caused  by  physiological  stasis 
of  bile  in  Texas  fever.     Note  the  fineness  of  the  capillary  network 
and  the  relation  of  the  capillaries  to  the  cells. 

120.  Glycogen.     Demonstration.     Liver   hardened    in    abso- 
lute alcohol  ;  paraffin  ;  sections  stained  with   a  solution   of   iodin  ; 
mounted  in   iodin-gum  arabic.     The  glycogen  is  stained  a  yellowish 
brown  ;  note  its  accumulation   in  one   end   of  the   cells  (generally 


57 
THE  RESPIRATORY  SYSTEM. 

LABORATORY    WORK    FOR 
References  : 

Points  for  Quiz  : 

° 
Laboratory  Report  .-OJUT>  cj-v^Ju*.  Q,^  t  \  Dn_ 


Go  to  the  delivery  desk  for  (a)  assigned  slide,  121  ;  (b)  paraf- 
fin section,  128  ;  (c)  colpdion  sections,  122,  123  ;  (d)  sections  ready 
to  mount,  124,  126  ;  (e)  prepare  125. 

(10)  Soft  palate.  Cat.  Assigned  for  study.  The  nasal 
side  of  the  soft  palate  will  afford  a  demonstration  of  the  ciliated  epi- 
thelium of  the  nasal  fossae.  This  will  be  spoken  of  in  your  text 
book  as  a  stratified  ciliated  epithelium.  Compare  it  with  the  epithe- 
lium in  the  trachea. 

LARYNX. 

121.  Larynx.  ~.     Assigned  for  study.     L/ongisec- 
tion.                          ;  collodion  ;  sections  /*.     Hematoxylin  and 
picrofuchsin.     Understand  what  structures  are  shown  in  the  section. 
Of  the  cartilages  shown  in  the  section,  the  first  is  the  hyoid,  the  sec- 
ond the  thyroid,  followed  by  the  cricoid  and  the  rings  of  the  trachea. 
Identify  the  true  and  false  vocal  cords  and  the  ventricle  of  the  larynx. 
Note  the  character  of  the  epithelium  in  the  different  regions,  i.  e., 
in  the  ventricle  and  upon  the  false  and  true  vocal  cords.     Are  glands 
present  ?     Where  are  they  located  ?     Note  also  the  structure  of  the 
mucous  membrane  and  its  variation  in  the  different  regions. 

TRACHEA. 

122.  Trachea.     Cat  (young).     Transection.     Picric  alcohol  ; 
collodion  ;  sections    "i  ij      p.     Stain   with   hematoxylin   and    picro- 
fuchsin. 

Identify  the  three  coats  of  the  trachea,  (/)  the  mucous  coat, 
(2)  the  submucous  coat,  (j)  the  fibrous  coat  with  its  incomplete 


58 

ring  of  cartilage,  studying  carefully  the  structure  of  each  coat. 
Compare  the  lining  epithelium  with  that  in  the  ventricle  of  the 
larynx  and  upon  the  soft  palate.  Note  especially  the  overlapping 
of  the  ends  of  the  cartilaginous  rings  and  the  character  of  the  epi- 
thelium in  the  fold  of  the  mucosa  caused  thereby,  and  the  relation 
of  the  muscle  to  the  cartilage.  In  the  last  respect,  compare  with 
the  relations  in  man  and  sheep,  as  demonstrated  to  you. 

123.  Trachea.     Cat    (old).     Transection.      Picric    alcohol; 
collodion  ;  sections  r.         //.     Stain   with    hematoxylin   and    picro- 
fuchsin. 

Recognize  in  this  preparation  exactly  the  same  structures  ident- 
ified in  122  and  compare  the  two  preparations,  especially  as  to  the 
epithelium  lining  the  fold  where  the  cartilages  overlap.  This  will 
illustrate  how  the  original  character  of  an  epithelium  is  altered  by 

changed  conditions. 

LUNG. 

124.  Lung.  Transection  (or  longisection)  of  a  lobe 
(in  part).     Picric  alcohol  ;  collodion  ;  sections  yu.     Stained  m 
to  to.  yu^ 

Examine  the  gross  preparations  of  lung,  showing  the  lobes, 
and  the  appearance  of  lung  tissue  seen  in  surface  view  and  in  sec- 
tion. Is  a  further  division  of  lobes  into  lobules  recognizable  ?  The 
section  will  illustrate  the  structure  of  normal  lung. 

The  following  features  are  to  be  noted  :  («)  the  general  struc- 
tural appearance  of  lung  tissue,  (£)  the  structure  of  a  bronchus  or 
bronchiole,  (e)  the  infundibula  with  air  sacs  opening  into  them,  and 
(oT),  if  possible,  the  transition  of  terminal  bronchus  or  alveolar  duct 
to  infundibulum. 

125.  Dried    lung.     Cat.     The  fresh  lung  was  inflated  and 
dried.     Prepare  a  slide  with  a  rubber  cell  (§  103,  b),  and  mount 
within  the  cell  two  sections  of  lung  cut  free-hand,  a  surface  section 
and  a  deep  section,  mounting  the  surface  section  pleural  side  up. 
Cover  and  seal  (§  103,  £). 

This  preparation  will  illustrate  well  the  infundibula  and  the 
compartments  in  their  walls,  the  air  sacs.  It  is  possible  that  there 
may  be  a  good  natural  injection  of  the  blood  capillaries  (filled  with 
blood, — yellow)  ;  if  so,  note  the  size  of  the  capillaries  and  the  dense- 
ness  of  the  net-work. 


59 

126.  Lung.  .     Injected  with  Berlin  blue  gelatin 
mass  (§  128)  ;  alcohols  ;  collodion  ;  sections  /^.     No  staining. 

Search  the  preparation  to  find  places  showing  well  the  capillary 
net-  work  in  the  walls  of  the  air  sacs,  and  compare  it  with  the  natural 
injection,  if  present,  in  125. 

127.  Respiratory  epithelium.    Cat.     Demonstration.    Free- 
hand section  of  silvered  lung.     The  blackened  cell  cement  outlines 
the  cells  lining  the  air  sacs,  of  which  recognize  the  two  kinds,  large 
flat  cells  with  irregular  outlines  and  small  granular  cells.  Understand 
the  significance  of  the  two  kinds. 

THYROID. 


128.  Thyroid.  £  2><^  ^  cr^  Chrome-oxalic  ;  paraffin  ;  sections 
<o  /*.  Stain  with  hematoxylin  and  eosin.  Consult  the  gross 
preparations  for  the  position,  shape  (general),  and  color  of  the 
glands  ;  their  appearance  superficially  and  in  section.  In  the  sec- 
tion there  are  to  be  noted,  the  shape,  size,  and  structure  of  the 
acini  ;  the  colloid  mass  ;  the  connective  tissue  dividing  the  gland 
into  lobes  and  lobules  ;  the  blood  vessels.  Study  well  the  epitheli- 
um in  connection  with  the  formation  of  the  colloid  mass.  Can  you 
recognize  two  kinds  of  cells  ?  This  is  a  ductless  gland  ;  in  what 
way  may  the  secretion  be  utilized  by  the  body  ? 


TOPICS    FOR    QUIZ— isth    Week 

THE  RESPIRATORY  SYSTEM 


1.  Constituents  of  the  respiratory  system. 

2.  Compare  the  respiratory  canal  of  the  nose  and  the  structure  of  the  oral 
cavity.  (Shown  in  the  section  of  the  soft  palate.) 

3.  Structure  of  the  larynx. 

4.  Structure  of  the  trachea.     What  does  the  trachea  merge  into  ? 

5.  Structure  of  the  lung  ? 

6.  Ivobules  or  infundibula,  and  air  cells  or  sacs. 

7.  Character  of  the  epithelium  in  different  parts  of  the  respiratory  tract. 

8.  Vascular  supply  of  the  lungs. 

9.  How  distinguish  a  transection  and  a  longisection  of  trachea  ? 
10.  Thyroid.     Where  is  it,  and  what  is  its  structure  and  fuftcbkm  ? 


6o 
THE  URINARY  ORGANS. 

LABORATORY   WORK    FOR   THE--^-- WEEK,    "i^-   S -I 

References:     \\sj<rJ&-     Ok. 
Points  for  Quiz  : 

Laboratory  Report :  ^^.w^JU^.      \  x  v-i  %"fctcxoo    t  -x.  n_  t\-^^»  i  •».  if 

rr-  w- 

Due:    •***•     ^ 

Obtain  from  the  delivery  desk  (a)  paraffin  sections  129,  132, 
i33»-  W  collodion  sections  130,  134,  135,  136,  137,  138. 

KIDNEY. 

129.  Kidney.  \W«X*A*.        Transection.      Miiller's  fluid  ;  par- 
affin ;  sections   0.6     ft.     Stain  with  hematoxylin  and  eosin. 

Kxamine  the  gross  preparations  of  kidney  for  (a)  the  form  and 
color  of  the  organ,  (£)  the  unilobular  (apparently)  and  multilobular 
kidney  ;  compare  the  kidney  of  the  child  with  that  of  the  adult, 
and  with  the  kidney  of  the  ox.  In  the  transected  and  longisected 
organs,  recognize  if  possible  (#)  the  cortex  (superficial  zone)  and 
(#)  medulla,  (c)  the  papillae.  Some  of  the  kidneys  have  one,  others 
several  papillae  ;  note  in  which  animals  each  of  these  conditions  oc- 
curs and  correlate  lobules  and  papillae. 

In  studying  the  section  recognize  the  following  regions  :  {a} 
cortex  and  (£)  medulla  ;  in  the  cortex,  medullary  rays  and  laby- 
rinth, and  in  the  medulla,  the  pyramids  and  the  columns  of  Bertini. 
Study  carefully  these  four  regions,  identifying  the  structures  that 
should  be  present  in  each.  Understand  the  course  the  uriniferous 
tubule  would  pursue  from  its  beginning  (Bowman's  capsule)  to  the 
opening  of  the  collecting  tubule  into  the  pelvis  of  the  kidney  and 
the  regions  in  which  its  successive  parts  are  found.  Again  examine 
the  longisected  kidneys  ;  recognize  the  regions  and  note  the  struct- 
ural appearance  of  each,  for  which  you  have  the  explanation  in  this 
preparation. 

130.  Injected    kidney.     Cat.      Transection.     Blood    vessels 
injected  with  carmine  gelatin   mass  ;  collodion  ;  sections     '  $ft    J*. 


6i 

This  preparation  will  illustrate  the  relations  of  the  blood  vessels 
within  the  kidney,  and  should  be  studied  in  connection  with  129, 
which  it  supplements.  Understand  the  regions  in  which  the  fol- 
lowing occur  and  the  courses  they  pursue  :  (a)  the  renal  arteries, 
(6)  "interlobular"  arteries,  (c)  afferent  arteries,  (d)  glomeruli,  (e) 
efferent  vessel,  (/)  capillaries,  (g)  interlobular  veins,  (/*)  renal 
veins,  and  (i)  the  arteriae  and  venae  rectae.  Identify  them  in  the 
preparation. 

131.  Bowman's    capsule.     Cat.     Demonstration.     Injected 
kidney  ;  paraffin  ;  sections          ~  /*.     Stained  with  hematoxylin  and 
picric  alcohol.     The  injection  has  caused  a  transudation  of  fluid  into 
Bowman's  capsule  ;  this  fluid  has  taken  the  hematoxylin  stain,  thus 
illustrating  the  relation  of  the  glomerulus  to  Bowman's  capsule  and 
the  beginning  of  the  urinary  tubule. 

132.  Papilla  of  the  kidney.     («)  Horse.     Transection. 
paraffin  ;    sections     /Q      IJL.       Stain   with    hematoxylin   and   picro- 
fuchsin.     From   your  study  of  129  and  130  identify  the  tubules  and 
vessels  shown  in  this  section  and  note  their  grouping  in  relation  to 
each  other. 

133.  Same  ;     (£)    Homo.     Miiller's    fluid  ;    paraffin  ;    sections 
/o      }*.     Stain  with  hematoxylin  and  eosin.     Compare  this  with 

132. 

URETER. 


134.  Ureter.  VH,  .  Transection.  Picric  alcohol  ;  col- 

lodion ;  sections  %_n  jn.  Stain  with  hematoxylin  and  picrofuchsin. 

In  addition  to  the  recognition  of  the  three  coats  and  the  struct- 
ure of  each,  note  well  (a)  the  character  of  the  lining  epithelium  and 
compare  it  with  the  stratified  squamous  epithelium  (10,  99),  and  (b) 
the  direction  of  the  muscular  fibers  in  the  two  (or  three)  muscular 
layers,  and  compare  with  the  condition  in  the  digestive  tract,  e.  g., 
intestine.  Compare  the  epithelium  of  the  ureter  with  that  of  the 
pelvis  of  the  kidney  (129). 

BI,  ADDER. 


1  35.  Bladder,—  distended.  C^Jk  .<VvvJkrr  O^UL  ^iJWfct  coll°- 
dion  ;  sections  X^  /*.  Stain  with  hematoxylin  and  picrofuchsin. 

Note  the  three  coats,  and  their  structure.  Can  you  recognize 
three  layers  in  the  muscular  coat  ?  What  is  the  course  of  the  fiber 


62 

bundles?  Recognize  blood  vessels,  nerves  (and  ganglia  ?).  Study 
well  the  epithelium  and  compare  it  with  the  epithelium  of  the  ureter. 

136.  Bladder, — collapsed.  VAX/A*.  JU*WX.  ;  (collodion  ) 
.  sections    10      jit.     Stain  with  hematoxylin  and   V«>S«J  ^^^^.This 

preparation  is  to  be  compared  with  135  for  (a)  the  epithelium,  its 
differences  in  a  distended  and  contracted  condition,  and  (£)  the  lay- 
ers of  the  muscular  coat. 

URETHRA. 

137.  Urethra — female  (and  vagina).     Cj^V         Transection. 
<V.^_AJ1jj^A/>^   collodion  ;  sections     1  u   /^-     Stain  with  hematoxylin 
and  picrofuchsin. 

Study  the  preparation  carefully,  noting  the  coats,  the  character 
of  the  lining  epithelium,  the  layers  composing  the  muscular  coat, 
and  the  direction  of  the  fibers  ;  compare  with  the  condition  in  the 
ureter  and  bladder.  Are  glands  present  ?  Note  the  blood  .vessels 
of  the  mucosa. 

138.  Urethra — male.       %-v><^     Transection.    "h**As>JW* 
collodion  ;    sections   'L^     j^.     Stain   with   hematoxylin   and    picro- 
fuchsin. 

Compare  this  preparation  with  137  and  note  differences.  What 
is  the  character  of  the  epithelium  and  therefore  what  part  of  the 
urethra  is  it  ?  Are  the  muscular  layers  well  denned  ? 


TOPICS    FOR    QUIZ— i6th   Week. 


THE  URINARY  ORGANS. 


r.  What  are  the  urinary  organs? 

2.  General  structure  of  a  kidney  as  seen  in  longisection  ? 

3.  Medullary  rays. 

4.  Urinary  tubule,  entire  extent  with  names  of  parts. 

5.  Vascular  system  of  the  kidney. 

6.  Malpighian  bodies  or  corpuscles.    Bowman's  capsule,  glomerulus. 

7.  Epithelium  of  the  urinary  tubules,  pelvis  of  the  kidney,  ureter,  bladder 
and  urethra. 

8.  Structure  of  the  urocyst  or  urinary  bladder  (a)  empty  and  (b)  distended. 

9.  Structure  of  the  ureter. 


63 
GENITAL  ORGANS  :    MALE. 

LABORATORY   WORK    FOR   THE WEEK. 

References : 
Points  for  Quiz  : 

Laboratory  Report  : 

Due  : 

Obtain  from  the  delivery  desk,  (a)  paraffin  sections,  141,  143  ; 
(£)  collodion  sections,  139,1  40,  144,  (c)  142,  cover-glass  preparation. 

TESTICLE. 

139.  Testis,  .     Longisection  ;  collodion; 
sections             ju.     Stain  with  hematoxylin  and  picrofuchsin. 

The  following  parts  should  be  recognized,  (a)  the  testis,  and 
(£)  the  epididymis  ;  in  the  testis,  the  three  coats,  the  lobules  of  the 
testis,  and,  composing  the  lobules,  the  seminiferous  tubules,  straight 
tubules  and  rete  testis  ;  in  the  epididymis,  note  its  relation  (as  a 
mass)  to  the  testis,  the  structure  of  the  tubules,  the  character  of  the 
epithelium.  Does  the  section  include  the  globus  major  or  minor  or 
both  ?  Is  the  vas  epididymis  sectioned  ?  Can  you  determine  the 
vas  deferens  ?  In  addition,  note  the  blood  vessels. 

140.  Testis,  .     Transection   of  the   testis   in   the 
tunica   vaginalis.                        ;    collodion  ;    sections  //.     Stain 
with  hematoxylin  and  eosin. 

Identify,  as  far  as  possible,  the  parts  recognized  in  139,  and  in 
addition,  the  tunica  vaginalis  with  its  two  layers,  and  the  mediasti- 
num. Note  the  position  occupied  by  the  blood  vessels.  What  is 
the  cavity  between  the  two  layers  of  the  tunica  vaginalis  and  its 
lining  epithelium  ?  Identify  the  vas  defefens. 

141.  Spermatogenesis.      Rat.      Section    of  a   part    of    the 
testis.  ;  paraffin  ;    sections  //.     Stain    with    hema- 
toxylin and  acid  fuchsin  (l/i%  solution). 

Examine  different  tubules  and  find  if  possible  five  different  stages 
in  the  formation  of  the  spermatozoa,  remembering  that  the  process 


64 

occurs  from  the  outside  toward  the  lumen  of  the  seminiferous 
tubules,  so  that  in  the  innermost  layer  would  be  found  maturing 
(or  mature)  spermatozoa,  in  the  outermost  layer  spermatogonia 
(parietal  cells),  and  between  spermatocytes  (mother  cells)  and 
spermatids  (daughter  cells),  which  metamorphose  into  the  sperma- 
tozoa. Find  also  the  supporting  cells  (Sertoli  cells)  and  observe 
their  relation  to  the  ripening  spermatozoa. 

142.  Spermatozoa.     Cat.     Dried  on   the   cover   glass.     No 
staining.     Mount  on  a  shellac  ring  (§  103,  a). 

Recognize  the  three  parts — head,  middle-piece,  and  tail,  noting 
the  shape  and  relative  length  of  each  ;  compare  with  the  sperma- 
tozoa found  in  the  tubules  and  ducts  (vasa)  in  139,  141,  noting 
the  staining  reactions  of  the  parts. 

VAS   DKFKRENS. 

143.  Vas     deferens.  .  ;   paraffin  ;     sec- 
tions            yu.     Stain  with  hematoxylin  and 

Determine  the  number  oi  the  coats  and  their  structure  ;  the 
course  of  the  fibers  in  the  muscular  layers  and  the  character  of  the 
epithelium,  and  compare  it  with  the  epithelium  found  in  the  tubules 
of  the  epididymis. 

PROSTATE    GLAND. 

144.  Prostate.  .     Transection  of  the  gland  and  ure- 
thra.                         ;  collodion  ;  sections  yu.     Stain  with  hema- 
toxylin and  eosin. 

Understand  the  relation  of  the  gland,  to  the  bladder  and  the  ure- 
thra. In  the  section,  observe  the  relation  of  the  gland  mass  to  the 
urethra.  Is  a  division  into  lobes  indicated  ?  Note  the  capsule  and 
its  structure,  the  acini  and  ducts  forming  the  gland  mass,  together 
with  connective  tissue  and  plain  muscle.  Does  the  section  include 
the  prostatic  sinus  or  the  ejaculatory  ducts  (vasa  deferentia)  ? 


65 
THE  GENITAL  ORGANS  :    FEMALE. 

LABORATORY   WORK    FOR    THE WEEK. 

References  : 
Points  for  Quiz  : 

Laboratory  Report : 

Due  : 

Obtain  from  the    delivery    desk,   (a)  paraffin  section,   147  ; 
(b)  collodion  sections,  145,  146,  148. 

OVARY. 

145.  Ovary.  .  ;  collodion  ;  sections  /*. 

Stain  with  hematoxylin  and  eosin. 

Examine  the  gross  preparations  for  the  shape,  superficial  ap- 
pearance of  the  ovary  and  its  relations  to  the  ligaments  and  to  the 
Fallopian  tube.  In  the  section,  note  the  regions,  roughly  defined,— 
the  cortex  and  medulla  and  the  structural  appearance  of  each.  As 
composing  the  ovary,  note  (a)  the  covering  tunica  albuginea,  (b) 
the  ovarian  stroma  containing  (V)  Graafian  follicles  at  various  stages 
of  maturity,  together  with  (d)  the  blood  vessels  and  nerves.  Points 
to  which  attention  is  called  are  (z)  the  peculiar  structure  of  the 
stroma  of  the  ovary,  (2}  the  large  size  of  the  veins,  and  (j)  the 
structure  of  the  mature  Graafian  follicle.  In  studying  the  Graafian 
follicle,  recognize  (<z)  the  theca  and  its  layers  ;  (£)  the  membrana 
granulosa  surrounding  the  cavity  of  the  follicle  and  bearing  on  one 
side  an  eminence  containing  the  ovum, — the  discus  proligerus. 

Study  carefully  the  structure  of  the  ovum  and  its  parts,  identi- 
fying it  as  a  single  cell,  and.  comparing  it  with  other  cells.  Under- 
stand how  the  ovum  may  escape  from  the  ovary  by  the  rupture  of 
the  Graafian  follicle.  Compare  i. 

(19).  Ovary.  Cat,  new-born  (1-2  weeks).  Assigned  for 
study. 

This  preparation  illustrates  especially  (a)  the  germinal  epi- 
thelium, (£)  the  egg-nests  or  egg-tubes,  and  (V)  early  stages  in  the 
formation  of  Graafian  follicles.  Examine  the  germinal  epithelium 
to  find  here  and  there  the  large  sexual  cells.  Can  you  find  inter- 


66 

mediate  steps  between  them  and  the  young  ova  ?  Remember  the 
difference  of  opinion  as  to  the  origin  of  the  follicle  cells. 

(18).  Ovary.  Cat  (young).  Preparation  previously  made 
(18). 

In  this  will  be  found  stages  in    the  formation  of  the  Graafian 

follicles.  Note  the  layer  of  young  ova  under  the  tunica  albuginea, 
Graafian  follicles  with  one  layer  of  follicle  cells  surrounding  the  ova, 
follicles  with  a  several-layered  follicular  epithelium,  and  older  follicles 
in  which  the  cavity  of  the  follicle  has  appeared. 

146.  Corpus  luteum.     Section  of  the  ovary  of  a  pregnant 
;  collodion  ;  sections  yu.     Stain  with 

In  studying  this  preparation  understand  when  and  how  the 
corpus  luteum  is  developed.  Note  its  structure,  observing  the  poly- 
hedral cells,  the  connective  tissue,  and  the  blood  vessels.  Is  there 
any  trace  of  the  original  cavity  of  the  follicle  ? 

FAI^OPIAN    TUBE. 

147.  Fallopian  tube.  .  ;  paraffin  ;  sec- 
tions            //.     Stain  with  hematoxylin  and  picrofuchsin. 

Identify  the  coats,  noting  their  structure,  the  extensive  folds  of 
the  mucosa,  the  character  of  the  lining  epithelium,  the  presence 
of  a  muscularis  mucosae  (?)  the  arrangement  of  the  fibers  in  the 
muscular  coat,  the  character  and  location  of  the  blood  vessels.  Are 
all  the  cells  of  the  epithelium  ciliated  ? 

UTERUS. 

148.  Uterus.  .  Transection.  ; 
collodion  ;  sections             /*.     Stain  with  hematoxylin  and  eosin. 

The  same  coats  are  present  as  in  147,  but  note  the  differences, 
especially  (#)  the  character  of  the  lining  epithelium,  (b)  the  pecu- 
liar structure  of  the  mucosa,  (c)  the  glands  contained  in  the  mucosa, 
(d)  the  thickness  of  the  innermost  muscular  layer  and  the  direction 
of  its  fibers,  (e)  the  veins. 

VAGINA. 

(I37)-.  Vagina.  .  Transection.  Preparation  already 

made  (137). 

Recognize  the  three  coats  and  study  the  structure  of  each,  not- 
ing (a)  the  character  of  the  lining  epithelium,  (#)  the  structure  of 
the  mucosa,  (c)  the  course  of  the  fibers  of  the  muscular  coat.  Are 
ganglia  recognizable  in  the  muscularis  or  mucosa  ?  Note 'the  num- 
ber and  size  of  the  blood  vessels. 


67 
THE  SKIN  AND  ITS  APPENDAGES. 

LABORATORY    WORK    FOR   THE  -My.-  WEEK, 
References  :     CJU  .  x  v    (**  ^^jL^oo^^^Ur  ^ 
Points  for  Quiz  : 

Laboratory  Report  :  ^&«^*    V^V  \  \>  T.  ,  \  vu  ,  \  ,b 

Due  : 


Obtain  from  the  delivery  desk  (a)  assigned  slides  152  and  154, 
(£)  paraffin  sections  150,  152,  157,  158  and  159,  (c}  collodion  sec- 
tions 149,  151,  156,  (d}  153  will  be  sectioned  in  your  presence  dur- 
ing the  week  ;  read  §§  61-63. 


THE   SKIN. 


149.  Skin  of  (palm  or  heel.)  Homo.     Vertical    section 

.  collodion  ;  sections  »  o  ju.  Stain  with  hematoxylin  and 
picrofuchsin.  t>'-«oo^c_ 

Identify  epidermis  and  corium  ;  in  the  former  recognize  the 
layers  and  study  their  structure  ;  are  the  layers  of  the  corium  dis- 
tinguishable ?  Search  the  preparation  carefully  for  sweat  glands  ; 
corpuscles  of  Meissner  may  be  present  in  the  papillae  of  the  corium. 
Are  inter-cellular  bridges  shown  in  the  stratum  Malpighii  ? 

(17).  Skin  of  palm  or  heel.  Homo.  Surface  section 
(slightly  oblique).  Preparation  already  made  (17).  In  this  prep- 
aration note  (0)  the  inter-cellular  bridges  of  the  cells  of  the  Mal- 
pighian  layer  and  the  relation  of  the  deepest  cells  next  the  corium 
to  the  connective  tissue,  (£)  the  transected  papillae  of  the  corium. 

150.  Skin,  general  body  surface.     Homo.     Vertical  section. 

;  paraffin  ;  sections  r~\  //.  Stain  with  hematoxylin 
and  picrofuchsin.  Study  this  preparation  carefully  in  comparison 
with  149,  identifying  the  epidermis  and  corium,  the  layers  compos- 
ing them,  and  the  relative  development  of  each.  Does  the  section 
show  the  presence  of  hairs  ?  Sweat  glands  ? 

151.  Lip.     Homo.     Transection.   SH^AOAA!/*  ;  collodion;  sec- 
tions    XO     JA.     Stain  with  hematoxylin  and  picrofuchsin. 


68 

This  preparation  affords  a  demonstration  of  the  transition  of  the 
epidermis  of  the  skin  to  the  epithelium  of  the  oral  mucosa.  Note 
well  the  character  of  the  epithelium  upon  the  two  sides  of  the  lip, 
and  the  presence  of  hairs  on  the  dermal  side.  Study  the  section 
also  for  the  structure  of  the  skin, — the  layers,  glands,  etc.  As  illus- 
trating the  structure  of  the  lip,  note  the  striated  muscle,  connective 
tissue,  nerves  and  blood  vessels  and  their  arrangement. 

152.  Lip.     Horse  or  calf.     Assigned  for  study.     Transection. 
;  collodion  ;  sections  //.     Stained  with 

Compare  this  preparation  with  151,  recognizing  the  features  enume- 
rated above.     Note  the  greater  development  of  the  hair  of  the  skin. 

NAII,. 

153.  Finger.     Child.     Transection  through  the  tip. 

frozen  section  (§  61).      Now  in   water.      Stain   with   hydrochloric 
acid  carmine  minutes  (§  87),  and  counter-stain  slightly  with 

picric  alcohol. 

This  preparation  illustrates  :  (#)  the  structure  of  the  nail,  (£) 
the  skin  (of  the  finger).  Note  well  the  structure  of  the  nail  and  the 
nail-bed  with  its  longitudinal  corrugations  cut  across  ;  the  relation 
of  nail  and  nail-bed  to  the  skin  of  the  finger.  In  addition  there  are 
illustrated  the  structure  of  Pacinian  corpuscles  and  sweat  glands. 

HAIR. 

154.  Hair.     Homo.     Assigned  for  study.     Section  of  scalp, 
cutting  the  hairs  at  right  angles.  ;  paraffin  ;  stained  with 
hematoxylin,  picrofuchsin,  and  methyl  green. 

Since  the  section  cuts  the  hairs  at  right  angles  and  the  hairs  are 
oblique  to  the  surface,  there  appear  transections  of  hairs  at  different 
levels.  Study  carefully  the  sections  of  hair,  identifying  the  layers 
composing  the  hair  and  follicle  at  different  levels,  especially  compar- 
ing sections  through  the  mouth  of  the  follicle,  through  the  middle  of 
the  root,  and  through  the  hair  bulb.  Study  the  structure  of  the 
sebaceous  glands,  their  relation  to  the  hair  follicle  and  the  point  at 
which  they  open. 

155.  Hair.     Homo.     Demonstration.     Section   of  scalp,   cut 
longitudinally  with  the  hair  showing  the  entire  length.     Note  care- 
fully the  root  of  the  hair,  the  follicle  and  its  relation  to  the  epider- 
mis, the  sebaceous  glands  and  the  hair  muscle. 


69 

156.  Hair  muscle.     (Arrector  pili).     Cat.   Section  of  the  skin 
from  the  dorsal  side  of  the  tail,  cutting  the  hairs  longitudinally. 
Picric  alcohol  ;  collodion  ;  sections    x  o    j*.     Stain  with  hematoxy- 
lin  and  picrofuchsin. 

This  preparation  will  illustrate  especially  the  hair  muscles  and 
their  relation  to  the  hair  follicles.  Study,  noting  their  relation  to 
the  follicles  and  sebaceous  glands  ;  their  large  size  in  this  prepara- 
tion. Understand  their  action  in  raising  the  hair.  Compare  154, 
identifying  in  it  the  hair  muscle. 

MAMMARY    GLAND. 

157.  Mammary  gland.     CQW.     In  lactation. 

paraffin  ;  sections     /  O     /^.     Stain  with  hematoxylin  and  eosin. 

Observe  carefully  the  general  structural  appearance  of  the 
gland,  noting  the  lobulation  and  the  secreting  acini.  Study  care- 
fully the  epithelium  forming  the  acini,  noting  the  character  of  the 
cells  and  their  appearance  and  indications  of  functional  activity. 
Note  also  the  secretion  (if  present)  in  the  lumen  of  the  acinus.  Com- 
pare with  158  and  159. 

158.  Mammary  gland.     Cow.     In  lactation.     i%osmicacid 
(§22);  paraffin;  sections     \o     /*•     No  staining. 

This  preparation  supplements  157.  The  osmic  acid  blackens 
the  fat  globules  which  in  157  were  dissolved  out.  Observe  the  num- 
ber and  size  of  the  globules  in  the  epithelial  cells  of  the  acini  and 
in  the  secretion  in  the  lumen  of  the  acinus. 

159.  Mammary  gland.     Cow.     Not  in  lactation  <iAwY.<jv/<d, . 
paraffin  ;  sections     *0      /*.     Stain  with  hematoxylin  and  eosin. 

This  section  is  to  be  compared  with  157,  the  identity  of  struc- 

TOPICS    FOR    QUIZ— i7th    Week. 

THE  SKIN  AND  ITS  APPENDAGES. 

1.  What  is  the  skin  and  what  its  appendages  ? 

2.  Compare  the  skin  of  the  palm  or  sole  with  that  of  the  general  surface 
of  the  body. 

3.  Hairs.     What  are  hairs,  where  are  they  present  and  where  absent  ? 

4.  Nails,  claws  and  hoofs.     In  what  animals?     Structure. 

5.  Glands  of  the  skin.     Relations  with  the  hairs. 

6.  Hair  muscle. 

7.  Section  of  a  lip.     This  shows  a  transition  from  skin  to  mucosa.     How 


70 
THE  CENTRAL  NERVOUS  SYSTEM. 

"W*. 
LABORATORY   WORK    FOR   THE-  J-£  —  WEEK.  *cj  - 


References  :       CA/v  .  >  vi     ^  .  T_^L,  ^V^*^  -t^)  ^  b  ou 
Points  for  Quiz: 
Laboratory  Report  : 


Go  to  the  delivery  desk  for  (#)  paraffin  section  160,  (b)  prepar- 
ations already  stained  161,  162,  164-168,  (V)  section  163  will  be 
given  you  some  time  during  the  first  period. 

MYEL  (SPINAL  CORD). 

1 60.  Cervical  my  el.  Transection.  votn  Rath's 

fluid  ;  paraffin  ;  sections  JA..  Stain  with  hematoxylin  2-3 

hours,  picric  alcohol  30  seconds  or  longer. 

Gross  anatomy  :  Examine  the  preparations  of  myel,  noting  the 
three  regions — cervical,  thoracic  and  lumbar ;  the  end  of  the  seg- 
ment of  myel,  observing  the  halves  separated  by  the  dorsal  septum 
and  the  ventral  fissure,  the  central  gray  matter,  cinerea,  and  the 
superficial,  ectal  alba  (white  matter)  ;  the  spinal  nerves  formed  by 
the  union  of  a  dorsal  and  a  ventral  root,  the  former  with  an  enlarge- 
ment— the  ganglion. 

In  the  section  recognize  (a*)  the  lateral  halves,  (£)  the  ventral 
fissure,  (c)  the  dorsal  septum,  (d}  the  central  cavity — myelocoele 
with  its  lining  epithelium — endyma.  In  the  cinerea  note  (/)  the 
rough  division  into  lateral  masses  connected  by  a  commissure 
surrounding  the  myelocoele,  (2)  the  dorsal  and  ventral  cornua.  In 
studying  the  structure  of  the  cinerea  note  (a)  the  general 
structural  appearance  of  the  substantia  spongiosa  and  substantia 
gelatinosa,  (£)  the  size  and  character  of  the  nerve  cells  in  the  ven- 
tral cornua,  in  the  dorsal  cornua,  and  intermediate  region.  Con- 
sider the  significance  of  the  large  cells  of  the  ventral  cornu.  In  the 
alba,  distinguish  the  dorsal  columns  and  the  roughly  defined  lateral 


and  ventral  columns  ;  remember  the  physiological  tracts,  indistin- 
guishable in  this  preparation,  that  compose  the  alba.  Are  the  points 
of  exit  of  the  dorsal  and  ventral  roots  of  the  spinal  nerve  shown  in 
the  section  ?  if  not,  compare  with  161  and  ,162. 

161.  Thoracic  myel.  .     Transection.  ; 
stained  in  toto  in                      ;  collodion  ;  sections             JJL. 

Compare  this  preparation  with  the  above,  160,  noting  (a)  the 
general  shape  of  the  myel  in  the  thoracic  region,  (£)  the  size  and 
shape  of  the  cornua  of  the  cinerea,  (c)  the  number  and  size  of  the 
nerve  cells  in  the  parts  of  the  cinerea,  especially  the  cells  in  Clarke's 
column.  Is  a  lateral  horn  well  defined  ?  Are  issuing  fibers  of  the 
dorsal  or  ventral  roots  shown  in  the  section  ? 

162.  Lumbar  myel,  Transection.  ;  col- 
lodion ;  stained  in  toto  in                  ;  collodion  ;  sections             JJL. 

Compare  with  160  and  161,  noting  differences  in  (#)  the  gen- 
eral shape  of  the  myel,  (b)  the  relative  extent  of  the  cinerea  and  the 
shape  of  the  cornua,  (^  the  relative  number  and  size  of  the  nerve 
cells  in  the  regions  of  the  cinerea,  (d)  the  obliquely  coursing  root 
bundles  of  the  lumbar  nerves,  constituting  the  cauda  equina. 

163.  Myel.  Transection.     Golgi's   rapid   method 
(§  141)  ;  collodion  ;  sections  ju.     No  further  treatment  ;  now 
in  clearer.     Mount  in  balsam- without  a  cover-glass. 

In  this  preparation  there  will  be  outlined  by  a  black  precipitate, 
one  or  more  large  nerve  cells  and  their  dendrites,  so  that  their  ex- 
tent and  branching  may  be  seen  better  than  in  160,  161  or  162,  with 
which  comparison  is  to  be  made.  In  addition,  there  will  probably 
be  stained,  neuroglia  cells,  and  many  neurites  running  in  the 

cinerea. 

THE  (MEDUIXA)  OBLONGATA. 

Gross  anatomy.  In  connection  with  the  study  of  the  follow- 
ing sections,  make  a  careful  examination  of  the  preparations  of 
the  brain.  Ascertain  the  location  of  the  oblongata  in  relation  to  the 
myel  and  the  remainder  of  the  brain.  Upon  the  ventral  aspect 
recognize  (/)  the  pyramids  with  an  indication  of  their  decussation, 
(»  the  olives  (olivary  bodies),  (j)  the  pons,  and  O)  the  cranial 
nerves  springing  from  this  portion, — the  Vth  to  the  Xllth.  Upon 
the  dorsal  aspect  with  the  membranous  roof  (metatela)  removed, 
there  should  be  noted,  (a),  the  nuclei  (enlargements)  of  the  gracile 


and  cuneate  funiculi,  (£)  the  restiforra  body.  Note  the  relation  of 
the  restiform  body  and  the  pons  to  the  peduncles  of  the  cerebellum. 
In  examining  the  following  sections,  whenever  one  of  these  struct- 
ures is  mentioned,  examine  the  gross  preparations  and  ascertain  the 
plane  of  the  section. 

164.  Decussation  of  the  pyramids.     Cat.     Transection  of 
the  oblongata  at   the   level  of  the  decussation  of    the    pyramids. 

3%  and  5,%  potassium  dichromate  ;  collodion  ;  Weigert's  hema- 
toxylin  stain  (§  137)  ;  sections  u.  Now  in  95%  alcohol. 

Clear  and  mount  in  balsam  (alkaline). 

Recall  the  positions  in  the  myel  occupied  by  the  following 
tracts  :  (/)  the  crossed  and  direct  pyramidal  tracts,  (2)  the  direct 
cerebellar,  (j)  the  funiculi  gracilis  and  cuneatus  composing  the 
dorsal  column.  In  this  section  will  be  seen  the  decussation  of  the 
fibers  that  will  constitute  the  crossed  pyramidal  tracts  in  the  myel 
from  their  position  on  the  ventral  side  (pyramids)  to  the  lateral 
column  of  the  myel.  Recognize  the  dorsal  and  ventral  columns, 
the  substantia  gelatinosa  Rolandi,  the  nucleus  gracilis.  Laterad  of 
the  cap  of  gelatinosa  Rolandi  is  the  ascending  (sensory)  root  of  the 
Vth  cranial  nerve  ;  ventrad  of  this,  the  fibers  of  the  direct  cerebellar 
tract. 

165.  Decussation  of  the  lemniscus.  Transection 
of  the  oblongata,  through  the  nuclei  gracilis  and  cuneatus  and  the 
pyramids  (caudal     part).    Weigert's  hematoxylin  method  (§137); 
now  in  95%  alcohol  ;  sections             yw.    Clear  and  mount  in  alkaline 
balsam. 

v 

At  this  level,  slightly  cephalad  of  153,  the  decussation  of  the 
pyramidal  tracts  has  not  yet  begun,  the  gracile  nucleus  has  en- 
larged and  the  cuneate  nucleus  has  appeared.  Recognize  these 
features  and,  in  addition,  the  decussation  of  fibers  from  these  nuclei 
to  form  the  lemniscus.  Note  the  pyramids,  the  diminished  funi- 
culus  gracilis  (what  has  become  of  the  fibers?);  the  dorsal  cornu 
and  substantia  gelatinosa  Rolandi  ;  can  you  identify  the  ventral 
cornu  ?  Note  increased  distinctness  of  the  ascending  root  of  the  Vth 
and  the  direct  cerebellar  tract.  The  nuclei  of  the  Xllth  and  Xth 
nerves  and  the  fibers  of  the  Xllth  passing  to  their  exit  may  also 
be  seen.  The  section  may  be  slightly  cephalad  or  caudad  of  the 
level  desired  for  this  preparation  ;  which  is  it  ? 


73 

166.  Olives.  Transection   of  the    oblongata 
through    the    olives.     Weigert's    hematoxylin    method.     Sections 

yw.  Now  in  alcohol.  Clear  and  mount  in  alkaline  balsam. 
Identify  in  this  section  the  tracts  already  recognized  :  O)  the 
pyramids,  (£)  the  lemniscus,  (<r)  the  direct  cerebellar  tract,  (d) 
ascending  root  of  the  Vth,  and  (<?)  the  nucleus  of  the  Xllth,  (/)  the 
cuneate  nucleus  (and  tract).  Has  the  gracile  nucleus  disappeared  ? 
Note  the  (dentate)  olivary  nuclei  causing  the  prominences  upon 
the  lateral  aspects  of  the  oblongata  (the  olives).  Are  the  fibers  of 
the  Xth  or  Xllth  nerves  shown  ?  Note  the  extent  of  the  cavity 
(metacoele)  ;  the  membranous  roof  (metatela)  has  been  removed. 

167.  Pons.  Transection  of  the  oblongata 
through  the  pons.     Weigert's  hematoxylin  method  ;  sections 

/t.     Now  in  alcohol.     Clear  and  mount  in  alkaline  balsam. 

Identify  in  the  section  (a)  the  pyramidal  tracts,  (£)  the  lem- 
niscus, (c)  the  ascending  root  of  the  Vth,  (d)  the  posterior  longi- 
tudinal fasciculus.  Does  the  section  pass  through  the  nuclei  of  the 
Vlth,  Vllth  or  Vlllth  nerves  t  Are  any  of  these  nerves  shown  t 
What  has  become  of  the  restiform  body  t  Note  well  the  fibers 
forming  the  mass  of  the  pons,  their  decussation,  relation  to  the 
medipeduncle  of  the  cerebellum  ;  the  nuclei  of  the  pons. 

MESKNCKPHAL. 

Gross  preparations.  In  the  region  cephalad  of  the  cerebel- 
lum and  pons, — the  mesencephal,  identify  upon  the  dorsal  aspect  the 
pregemina  and  postgernina  (corpora  quadrigemina,  anterior  and  pos- 
terior); upon  the  ventral  aspect,  the  crura  (cerebri)  and  the  Hid 
cranial  nerve. 

1 68.  Crura  and  Gemina  (Pre- or  Post-).  .     Tran- 
section through  the  mesencephal.     Weigert's  hematoxylin  method  ; 
sections             // ;    now   in   alcohol.     Clear  and  mount  in  alkaline 

balsam. 

In  this  section  identify  (a)  the  pyramidal  tracts  (the  crusta), 

(3)  the  fillet,  (<:)  the  posterior  longitudinal  fasciculus.  Note  also 
the  central  cavity  (mesocoele)  with  the  lining  endyma,  the  central 
cinerea  surrounding  it,  the  nucleus  of  the  Hid  nerve.  Locate  the 
following  parts:  (/)  the  crusta,  (2)  the  substantia  nigra,  (j)  the 
tegmentum,  (4}  the  prepeduncles  of  the  cerebellum,  or  their  decus- 
sation. Does  the  section  include  the  red  nucleus?  Is  the  Hid 
nerve  shown  ? 


74 
THE  CENTRAL  NERVOUS  SYSTEM. 

LABORATORY    WORK    FOR   THE--'-^--  WEHK. 

References:      IV  .  >i/i    x,^  ^^y^.U 
Points  for  Quiz  : 

Laboratory  Report  : 

Due  : 


Go  to  the  delivery  desk  for  (a)  assigned  slide  177  ;  (b)  par- 
affin sections  172,  176;  (c)  sections  already  stained,  169,  170,  174, 
I75  >  (*O  I7I  an<i  J73  will  De  given  you  during  the  first  period. 


CEREBELLUM. 

Gross  Preparations.  Examine  the  gross  preparations,  noting 
(a)  the  parts  of  the  cerebellum  —  the  vermis,  lateral  hemispheres, 
and  the  flocculus,  comparing  man  and  the  different  animals  shown  ; 
(£)  the  lodes  of  the  cerebellum  and  the  folia,  their  size  and  direction  ; 
(£•)  the  three  peduncles  of  the  cerebellum  by  which  it  is  connected 
with  the  rest  of  the  brain  and  myel. 

169.  Cerebellum.     Cat.     Sagittal    section    of    the    vermis  ; 
vom  Rath's  fluid  ;  stained  in   toto  in'  paracarmine  ;  collodion  ;  sec- 
tions JJL. 

Note  the  deep  division  into  lobes  and  these  into  folia.  As  con- 
stituting the  cerebellum,  recognize  (a)  the  central  core  of  alba 
(fibers)  extending  into  the  lobes  and  folia,  (b)  the  covering  cinerea 
—  cortex.  Observe  the  layers  of  the  cortex,  the  grouping  of  the 
cells  of  the  granular  layer,  the  size  of  the  Purkinje  cells,  the  rela- 
tively small  number  of  cells  in  the  molecular  layer.  Remember  the 
real  extent  of  the  dendrites  of  the  Purkinje  cells,  the  destination  of 
the  neurites  of  the  Purkinje  cells  and  granules,  and  compare 
with  171. 

170.  Cerebellum.  .     Transection  (in  part)  ;  Wei- 
gert's   hematoxylin    method  ;   collodion  ;  sections  /^  ;  now  in 
95%  alcohol.     Clear  and  mount  in  alkaline  balsam. 

This  preparation  will  illustrate  the  ento-cinerea  of  the  cerebel- 
lum —  the  nucleus  dentatus  and  the  nuclei  of  the  roof,  as  well  as  the 


75 

commissural  fibers  of  the  cerebellum.  Note  the  shape  of  the  dentate 
nucleus  and  compare  with  the  nucleus  of  the  olive  (166).  Can  you 
identify  the  fibers  of  the  pre-  and  post-peduncles  of  the  cerebellum  ? 
Compare  with  169  for  the  general  structure  of  the  cerebellum. 

171.  Cerebellum.      Cat     (young).      Golgi's    rapid    method 
(§141)  ;  collodion  ;  sections  //,  cutting  the  folia  transversely. 

This  preparation  will  afford  a  demonstration  of  the  extent  of  the 
dendrites  of  the  Purkinje  cells,  the  basket  cells  of  the  molecular 
lay^er,  and  possibly  the  dendrites  and  neurites  of  the  granules  of  the 
granular  layer,  neuroglia  cells,  and  nerve  fibers. 

CEREBRUM. 

Gross  anatomy.  Consult  the  preparations,  studying  them 
carefully  so  that  you  may  have  clearly  in  mind  (a)  the  relative  size 
and  position  of  the  cerebrum,  (£)  that  it  consists  of  two  halves  con- 
nected with  each  other  (principally)  by  a  large  commissure,  the  cal- 
losum,  (c)  the  incision  of  its  surface  by  deep  depressions,  fissures, 
dividing  it  into  gyres,  {d}  the  aspects  of  the  cerebrum-  dorsal, 
frontal,  occipital,  mesal,  temporal.  The  cinerea  of  the  cerebrum 
comprises  (/)  the  superficial  cortex  and  the  hippocamp,  (2)  masses 
deeply  situated,  of  which  the  most  important  are  the  caudatum,  and 
lenticula,  with  which  may  be  considered  the  closely  associated  thala- 
mus  and  subthalamus.  Examine  the  series  of  sections  through  the 
cerebrum,  noting  the  cortex  and  the  location  of  these  masses  of  cine- 
rea, their  relation  to  each  other  and  to  the  cavities  of  the  diencephal 
and  cerebrum. 

172.  Cerebral  cortex.     Cat.     Transection  of  a  portion  at  the 
dorso-mesal  angle,  including  the  callosum  ;  95%  alcohol  ;  paraffin  ; 
sections  /*.     Stain  with  hematoxylin  (2-3  hours)  and  picro- 
fuchsin. 

The  points  to  be  noted  are  (a)  the  transected  gyres  with  the 
septums  of  pia  in  the  fissures,  (b)  the  structure  of  the  cortex  upon 
the  summit  of  a  gyre  and  at  the  bottom  of  a  fissure.  Can  you  recog- 
nize the  (four)  ill-defined  layers  ?  Note  the  number,  shape  and  size 
of  the  nerve  cells,  and  compare  with  71  and  173. 

173.  Cerebral  cortex.     Cat  (young).     Golgi's  rapid  method 
(§  141)  ;  collodion  ;  sections  ^  ;  in  clearer.     Mount  in  balsam 
without  a  cover-glass. 


76 

This  preparation  will  demonstrate  the  real  form  of  the  cells 
already  seen  in  172.  Find  good  examples  of  the  small  pyramidal 
and  large  pyramidal  cells,  and  note  the  extent  of  the  apical  process 
and  the  number  and  length  of  the  dendrites  arising  from  it  and  from 
the  cell  body.  Trace  the  neurite  as  far  as  possible.  Can  you  find 
collaterals  arising  from  it  ?  Find  also  good  examples  of  well  impreg- 
nated cells  from  the  layer  of  polymorphous  cells.  Doubtless  glia 
(spider)  cells  will  also  be  stained.  Compare  preparation  172. 

174.  Hippocamp.     Cat.     Transection  of  the  hippocamp  and 
adjacent  parts.     Weigert's  hematoxylin  method  ;    collodion  ;    sec- 
tions }A.     Now  in  95%  alcohol.     Clear  and  mount  in  balsam. 

The  parts  to  be  recognized  in  this  preparation  are  (z)  the  hip- 
pocampal  gyre,  (2)  hippocampus,  (j)  dentate  gyre,  (^)  fimbria, 
(5)  the  rima  with  the  plexus,  (6)  the  caudatum.  Note  the  con- 
tinuation of  the  cortex  of  the  hippocampal  gyre  as  the  grey  matter 
of  the  hippocamp  and  dentate  gyre  ;  observe  the  massing  of  large 
pyramidal  cells  in  the  hippocamp  and  the  dense  layer  of  small  pyra- 
midal cells  in  the  dentate  gyre,  the  continuation  of  the  fimbria  with 
the  central  (sub-cortical)  alba.  Recognize  that  the  hippocamp  is 
an  infolding  of  the  cortex. 

175.  Caudatum,   Lenticula,   and   Thalamus.     Transection 
through  a  hemicerebrum  (or  part)  including  these  masses  of  cinerea 
(ento-cinerea).     Weigert's  hematoxylin  method  ;  sections  yu. 

Now  in  alcohol.     Clear  and  mount  in  alkaline  balsam. 
i 

Locate  in  this  preparation  the  cortex,  the  caudatum,  lenticula, 
claustrum  and  thalamus  ;  between  the  claustrum  and  the  lenticula 
the  external  capsule  ;  between  the  lenticula  and  thalamus  (and  cau- 
datum) the  internal  capsule.  The  internal  capsule  is  the  continua- 
tion of  the  crusta  (168)  ;  the  subthalmic  region  is  continuous  with 
the  tegmentum  ;  the  thalamus  and  subthalamus  constitute  the  larger 
portion  of  the  diencephal,  intermediate  between  the  mesencephal 
(168)  and  the  cerebrum  (prosencephal).  Consult  the  transections 
of  brain  and  the  special  references  given. 

176.  Olfactory   bulbs.     Rabbit.      Transection  ;  vom   Rath's 
fluid  ;  paraffin  ;  sections  yu.     Stain  with  hematoxylin  ( i  hour) 
and  picrofuchsin. 

Recognize  the  following  layers  :  ( i )  the  layer  of  olfactory 
nerve  fibers,  (2)  the  layer  of  glomerules,  (3)  the  large  nerve  cells 


77 


(mitral  cells),  (4)  the  granular  layer,  (5)  the  olfactory  tract,  (6) 
the  entocinerea,  (7)  the  central  cavity  (rhinocoele)  with  its  lining 
endyma.  Understand  the  course  of  the  olfactory  impulse  from  the 
olfactory  nerve  fibers  to  the  olfactory  tract  (leading  to  other  parts  of 
the  brain).  Remember  that  in  the  human  brain  there  is  no  rhino- 
coele (in  the  adult). 


ADRENAL  BODY. 

177.  Adrenal  body.  Cat.  Assigned  for  study.  Chrome- 
oxalic  ;  paraffin.  Stained  with  hematoxylin  and  picrofuchsin. 

One  of  the  "  ductless  "  glands.  In  studying  this  preparation, 
note  (a)  the  regions, — cortex  and  medulla,  (£)  the  zones  of  the  cor- 
tex and  their  structure,  (<:)  the  structure  of  the  medulla  and  the 
number  and  character  of  the  contained  blood  vessels.  Other  duct- 
less glands  studied  by  you  are  the  thyroid  (128),  and  the  thymus 
(94).  Note  the  diversity  in  structure  and  origin  of  these  three 
glands. 

It  should  be  recognized  that  the  inclusion  of  the  adrenal  body 
with  the  organs  of  the  central  nervous  system  is  a  matter  of  con- 
venience rather  than  because  it  may  logically  be  grouped  with  them. 
On  an  embryological  basis,  however,  it  might  be  placed  in  the  peri- 
pheral nervous  system.  The  thyroid  (128),  likewise,  should  only 
be  grouped  with  the  respiratory  organs,  in  a  classification  the  basis 
of  which  is  embryology. 


78 
THE  ORGANS  OF  SPECIAL  SENSE. 

LABORATORY    WORK    FOR   THE  _?•-£.--  WEEK.    1/**M.  5  --«  0 

References  :      ^K  -  AVI  i 
£>~ 

Points  for  Quiz  : 

Laboratory  Report  : 


Obtain  from  the  delivery  desk,  (a)  paraffin  sections,  178,  179, 
180,  183,  185  ;  (b)  collodion  sections,  181,  182,  184. 

EYE. 

178.  Eye.     Pig.     Section  through  the  anterior  quarter  of  the 
eye-ball.     Zenker's  fluid  ;    paraffin  ;   sections  //.     Stain  with 
hematoxylin  and  picrofuchsin  . 

This  section  will  include  cornea,  iris,  ciliary  body  (muscle  and 
processes),  schlera,  choroid  and  retina.  Study  well  the  structure  of 
each  of  these  parts,  observing  the  relation  of  («)  schlera  and  cornea, 
(£)  iris  and  choroid,  (c}  ciliary  and  sensory  parts  of  the  retina. 
Note  also  the  canal  of  Schlemm,  ciliary  arteries,  sphincter  of  the 
pupil.  Is  there  any  trace  of  the  suspensory  ligament  of  the  lens  ? 

179.  Eye.     Cat.     Section  through  the  posterior  half  showing 
the  entrance  of  the  optic  nerve.     Zenker's  fluid  ;  paraffin  ;  sections 

/^.     Stain  with  hematoxylin  and  picrofuchsin. 

This  preparation  is  to  illustrate  the  structure  of  the  three  coats 
of  the  eye,  the  layers  of  the  retina,  and  the  relation  of  the  optic  nerve 
to  the  eye-ball,  especially  to  the  retina,  and  should  be  studied  to 
afford  demonstration  of  these  points. 

1  80.  Retina.  Pig.  Vertical  section.  ;  paraffin  ; 

sections  yw.  Stain  with 

In  this  section  identify  the  layers  of  the  retina  already  seen  in 
178  ;  be  sure  you  understand  the  real  structure  of  the  retina  as  re- 
vealed by  other  methods,  and  the  meaning  of  the  layers  brought 
out  by  ordinary  methods.  Distinguish  the  rods  and  the  cones  ;  re- 
member which  side  of  the  retina  faces  outward,  i.  e.,  is  toward  the 
other  coats  of  the  eye.  What  course  would  the  sensory  impulse 
pursue  in  order  to  reach  the  optic  nerve  ? 


79 

(27).  Cornea.  Cat.  Assigned  for  study.  Silvered  prepar- 
ation. Again  examine  the  preparation  of  cornea  already  studied, 
and  in  connection  with  the  structure  of  the  cornea  (177)  note  the 
cell  spaces  and  their  relation  to  each  other. 

181.  Optic   nerve.  Transection.     vom  Rath's  fluid; 
collodion  ;  sections         //.    Stain  with  hematoxylin  and  picrofuchsin. 

Note  the  connective  tissue  sheath  and  its  relation  to  the  schlera 
(178),  the  fibers  in  bundles.  Compare  the  individual  fibers  with 
the  fibers  of  the  peripheral  nerve  given  (74)  ;  what  is  the  differ- 
ence ?  Can  you  ascertain  ?  Are  the  retinal  vessels  included  ? 

182.  Eye-lids.  Transection.  collo- 
dion ;  sections             /*.     Stain  with  hematoxylin  and  eosin. 

In  this  section  recognize  (a)  the  dermal  and  conjunctival  sides 
and  the  character  of  the  epithelium  of  each,  (£)  the  cilia  (eye- 
lashes), (c}  the  Meibomian  gland  and  compare  with  the  sebaceous 
glands  of  the  hair  follicle  (154)  ;  (W)  the  orbicularis  muscle.  Can 
you  find  the  glands  of  Moll  ? 

183.  Lachrymal  Gland.  .  ;  ; 
. ;  sections               /*.     Stain  with  hematoxylin  and  eosin. 

Determine  the  type  of  gland,   noting  the  secreting  acini  and  the 
ducts  ;  and  compare  with  the  salivary  glands  (112,  113). 

THE   NOSE. 

184.  Nasal  fossae,  .     Transection  through  the  head 
and  nose.                  ;  decalcified  (§  131)  ;  collodion  ;  sections  //. 
Stain  with  hematoxylin  and  picrofuchsin. 

The  preparation  illustrates  (a)  the  respiratory  and  olfactory 
(sensory)  regions  in  the  nasal  fossae  of  this  animal  and  their  epi- 
thelium, (£)  the  nasal  septum  and  the  turbinal  bones. 

185.  Olfactory  mucous  membrane.     Guineapig.     Transec- 
tion of  a  part  of  the  more  dorsal  turbinal  bones.     Flemming's  fluid  ; 
stained  in  toto  ;  paraffin  ;  sections  /t. 

The  general  structure  of  the  sensory  epithelium  is  shown,  the 
glands  of  Bowman,  and  the  olfactory  fibers  in  the  mucosa.  Can  you 
distinguish  the  sustentacular  and  sensory  cells  in  the  epithelium  ? 


8o 
THE  ORGANS  OF  SPECIAL  SENSE. 

LABORATORY   WORK    FOR   THE-?*-'  ___  WEEK.      Vw«-*x  /  -*    ,/ 


References  :    <sJ^  -XVIM        *  t  V 

^Xx*     **  \  b  U- 

Points  for  Quiz  : 


Laboratory  Report  :  .  _ 


Obtain  from  the  delivery  desk  (a)  paraffin  sections  187,  188, 
(b)  collodion  section  186. 

THE  EAR. 

186.  Middle  ear,  inner  ear,  semi-circular  canals. 

;  collodion  ;  sections  yw.     Stain  with 

hematoxylin  and  eosin. 

Study  this  preparation  for  (a)  the  mucosa  of  the  tympanum 
and  the  character  of  the  epithelium,  (£)  the  bony  labyrinth,  mem- 
branous labyrinth,  perilymphatic  space  of  the  inner  ear.  Does  the 
section  include  one  of  the  maculae  or  cristae  of  the  membranous 
labyrinth?  Is  the  utricle  sectioned  as  well  as  the  semicircular 
canals  ?  Note  carefully  the  structure  and  relations  of  the  parts. 

187.  Cochlea.     Guinea-pig.     Longisection.     Zenker's  fluid  ; 
decalcified  (§131)  ;  paraffin  ;  sections  ^.     Stain  with  hema- 
toxylin and  eosin. 

Study  this  preparation  in  connection  with  figures  in  your  text 
book,  identifying  the  parts  and  studying  their  structure.  Is  Reiss- 
ner's  membrane  intact? 

THE   TASTE    BUDS. 

188.  Papilla  foliata.    Rabbit.    Longisection.    Zenker's  fluid  ; 
paraffin  ;  sections  //.     Stain  with  hematoxylin  and  eosin. 

The  taste  buds  will  be  found  in  the  epithelium  in  the  depres- 
sions or  transverse  fissures  of  the  papilla.  Examine  them  carefully, 
observing  their  form  and  structure,  relation  to  the  surface,  and  the 
gustatory  hairs. 


TECHNIC. 


§  i .  Very  few  structures  of  the  animal  body  can  be  examined 
microscopically  without  being  first  subjected  to  a  preparatory  treat- 
ment involving  in  most  cases  the  employment  of  complicated  meth- 
ods. Increase  in  our  knowledge  of  the  finer  structure  of  the  body 
in  the  past  has  been  accompanied  and  made  possible  by  the  improve- 
ments in  the  methods  employed.  Advance  in  the  future,  likewise, 
will  be  dependent  on  the  application  of  a  more  exact  technic.  For 
those  who  aim  to  do  work  in  practical  histology  and  pathology,  a 
mastery  of  the  more  important  methods  is  indispensible  ;  for  the 
student  of  pathology,  also,  very  desirable  is  the  acquisition  of  skill 
in  the  application  of  simple  methods  which  require  neither  expensive 
apparatus  nor  expenditure  of  time, — methods  which,  while  they  do 
not  advance  knowledge,  serve  to  meet  the  needs  of  a  rapid  clinical 
diagnosis. 

Of  the  multitudinous  methods  employed  in  microscopic  work, 
only  those  are  here  given  which  are  necessary  for  a  general  working 
knowledge  in  histology,  or  are  used  in  this  course.  Animal  tissues 
are  bulky  and  unfitted  for  examination  under  high  powers  of  the 
microscope  ;  examination  may  be  made  possible  in  one  of  two 
ways, — the  elements  composing  the  structure  may  be  separated  from 
each  other,  or  thin  slices  may  be  prepared.  Furthermore,  tissue 
may  be  examined  in  either  of  these  ways,  fresh  (or  alive),  or  after 
special  chemical  treatment.  The  technic  of  histology  involves 
then  : 

A.  Examining  fresh,  by  either  B.  or  C.     Advantageous  or  neces- 

sary when  haste  is  required,  or  in  examining  the   tissue 
alive. 

B.  Isolation  or  Dissociation.     Separating  out  the  elements  compos- 

ing a  tissue  by  (a)  teasing  or  (b)  treatment  with   reagents 
and  teasing. 

C.  Cutting  thin  sections  of  the  tissue  or  organ. 


82 

For  C.  are  generally  necessary  : 

1.  Fixing  the  tissue  (§§  18-38).     Hardening. 

2.  Sectioning  by  one  of  the  following  methods  : 

(a)  Free-hand,  without  an  imbedding  mass,  or 

(b)  With  an  imbedding  mass,  as 

(1)  By  the  Paraffin  method  (§  42),  or 

(2)  By  the  Collodion  method  (§  50),  or 

(3)  By  the  Freezing  method  (§  61). 

ISOLATION. 

§  2.  One  of  the  simplest  ways  of  examining  the  structure  of 
a  tissue  is  the  separation  from  one  another  of  the  structural  elements 
composing  it,  thus  permitting  its  analysis.  Likewise,  for  a  correct 
conception  of  the  forms  of  the  cells  and  fibers  of  the  various  tissues 
of  the  body,  one  must  see  these  elements  isolated  and  thus  be  able 
to  inspect  them  from  all  sides.  It  frequently  occurs  also  that  isola- 
tion is  not  quite  complete  and  one  can  see  in  the  clearest  manner 
the  relations  of  the  cells  or  fibers  to  one  another. 

In  the  employment  of  this  method  the  tissue  may  be  taken 
fresh  and  isolation  accomplished  by  teasing  with  needles  or  similar 
instruments  ;  or  it  may  be  treated  with  media  which  will  serve  to 
render  teasing  partially  or  entirely  unnecessary.  In  such  cases 
simply  shaking  or  gently  tapping  the  preparation  will  often  suffice. 
In  many  instances  it  is  desired  to  examine  the  tissue  while  the 
elements  are  still  alive,  as,  for  example,  in  the  study  of  ciliated 
cells,  and  recourse  must  be  had  to  some  "normal,"  "indifferent" 
medium.  Best  of  all  is  the  medium  with  which  they  are  bathed  dur- 
ing life — in  the  case  of  tissue  from  the  animal  body,  blood  serum, 
the  aqueous  humor  of  the  eye,  liquor  amnioticus,  or,  as  an  artificial 
substitute  often  more  convenient  if  reagents  are  to  be  used  subse- 
quently, normal  salt  solution,  being  a  iV~A^  solution  of  common 
salt  (sodium  chlorid)  in  distilled  water. 

The  chemical  agents  or  solutions  for  isolating  are,  in  general, 
the  same  as  those  used  for  fixing  and  hardening.  But  the  solutions 
are  only  about  one-tenth  as  strong  as  for  fixing  and  the  action  is  very 
much  weaker  and  requires  from  one  or  two  hours  to  as  many  days. 
In  the  weak  solution  the  cell  cement  or  connective  tissue  is  softened 


83 

so  that  cells  and  fibers  may  be  separated  from  one  another,  and  at 
the  same  time  the  cells  are  preserved.  In  other  words,  a  weak  fix- 
ing action  is  retained  while  the  hardening  action  is  reduced  on  dilu- 
tion. The  time  required  for  the  action  of  the  dissociator  varies  in- 
versely as  the  vehemence  of  the  fixer  and  the  density  of  the  tissue, 
2-3  hours  to  several  days.  In  fixing  and  hardening,  on  the  other 
hand,  the  cell  cement,  like  the  other  parts  of  the  tissue,  is  made 
firmer.  It  is  better  also  to  dilute  the  fixing  agents  with  normal 
salt  solution  than  merely  with  water. 

§  3.  Of  the  many  dissociators,  the  following  may  serve  most 
of  the  needs  of  histology  :  (i)  Miiller's  fluid  dissociator  ;  (2)  for- 
maldehyde dissociator  ;  (3)  nitric  acid  dissociator  ;  (4)  caustic  pot- 
ash dissociator  ;  (5)  Ranvier's  one-third  alcohol  ;  (6)  osmic  acid 

(A*). 

§4.  Miiller's  fluid  dissociator.  Formula:  Miiller's  fluid, 
i  part  ;  normal  salt  solution,  9  parts  (i.  e.,  potassium  dichromate, 
2.5  grams;  sodium  sulphate,  i  gram;  sodium  chlorid,  6  grams; 
water,  1,000  c.c.). 

This  is  a  good  general  dissociator  for  epithelia,  including  glands. 
Dilution  decreases  the  hardening  action  of  Miiller's  fluid  as  is 
shown  especially  on  the  cell-cement, — hence  its  dissociating  ac- 
tion. Considerable  latitude  in  time  is  allowed  in  the  use  of  this 
dissociator  ;  12  hours  being  often  sufficient,  although  a  stay  of  sev- 
eral days  in  the  dissociator  usually  does  no  harm. 

§  5.  Directions  for  use.  In  the  employment  of  this  fluid  for 
the  isolation  of  epithelial  cells,  proceed  as  follows  : 

Place  the  tissue  covered  with  the  epithelium  which  it  is  desired 
to  isolate  in  the  dissociator  in  a  shell  vial  or  dish,  where  it  may 
remain  from  2-3  hours  to  2-3  days  ;  for  the  epithelium  of  the  tra- 
chea, intestines,  etc.,  the  action  is  sufficient  in  2-3  hours,  although 
good  preparations  may  be  obtained  after  two  days  or  more.  For  the 
stratified  epithelia,  like  those  of  the  skin,  mouth,  etc.,  it  may  require 
1-3  days  for  the  most  satisfactory  preparations.  After  the  tissue  has 
remained  in  the  dissociator  a  sufficient  time,  scrape  the  epithelial 
surface  gently  with  a  scalpel  and  place  the  scrapings  on  a  slide  in  a 
drop  of  the  dissociator  ;  cover  and  examine.  If  one  proceeds  after 
two  hours  or  so,  probably  most  of  the  cells  will  cling  together,  and 


84 

in  the  various  clumps  will  appear  cells  on  end  showing  the  tops  or 
bases,  and  other  clumps  will  show  the  cells  in  profile. 

Tap  the  cover  gently  with  a  needle-holder  or  other  light  object 
in  order  to  separate  the  cells  from  each  other  more  completely. 
Many  fully  isolated  cells  as  well  as  cells  in  groups  will  be  seen. 
Examine  carefully. 

§  6.  Staining.  Scrape  gently  the  epithelial  surface  in  a  fresh 
spot  and  place  the  scrapings  on  the  slide  in  a  drop  of  eosin  (§  94)  or 
a  mixture  of  eosin  and  methylgreen  (§  92).  Mix  well  so  that  the 
stain  can  penetrate.  If  for  temporary  examination,  cover  immedi- 
ately and  examine  as  before. 

§  7.  Permanent  preparations.  If  a  permanent  mount  is  de- 
sired, (a)  allow  the  above  mixture  to  stand  for  5-10  minutes  ;  drain 
off  the  stain,  collecting  the  fragments  carefully  at  one  side  ;  add  a 
small  drop  of  glycerin  or  glycerin-jelly  (§§  104,  105)  ;  cover  and 
examine,  tapping  the  cover  gently  as  before,  if  necessary,  in  order 
to  separate  the  cells  more  thoroughly.  Unstained  preparations  may 
be  mounted  in  the  same  manner. 

(£)  The  following  method  will  also  give  satisfactory  stained 
preparations.  Place  a  small  mass  of  cells  in  a  drop  of  alum  carmine 
and  eosin  glycerin  (§  156)  ;  cover  and  isolate  the  cells  by  tapping 
gently  on  the  cover-glass  as  before.  Clean  and  seal  the  preparation 
(§  no). 

If  it  is  desired  to  make  a  permanent  mount  of  a  preparation 
already  covered  and  examined,  place  a  drop  of  glycerin  at  the  edge 
of  the  cover,  apply  to  the  opposite  side  of  the  cover  a  piece  of  blot- 
ting paper  or  filter  paper,  and  by  absorbing  the  fluid — dissociator  or 
stain — under  the  cover,  cause  the  glycerin  to  flow  under  to  take  its 
place. 

Seal  the  preparation  (§  no)  ;  it  is  usually  better,  however,  to 
allow  a  glycerin  or  glycerin-jelly  mount  to  stand  for  a  day,  being 
careful  that  it  is  not  touched,  and  then  seal. 

§  8.  Formaldehyde  Dissociator.  Formula :  40%  formal- 
dehyde (formalin),  2  c.  c. ;  normal  salt  solution,  i,oooc.  c.  (i.  e., 
.08%  sol.  of  formaldehyde  in  normal  salt  solution).  This  is  a  good 
general  dissociator  and  as  such  maybe  employed  instead  of  Miiller's 
fluid  dissociator.  It  is  especially  serviceable  in  the  isolation  of  the 
nerve  cells  of  the  brain  and  spinal  cord. 


§5 

§  9.  Directions  for  use.  Employ  this  fluid  for  the  isolation  of 
the  nerve  cells  of  the  spinal  cord  and  of  the  cerebral  cortex,  pro- 
ceeding as  follows  : 

Split  the  spinal  cord  along  its  median  plane,  separating  thus 
the  two  halves,  and  place  it  in  an  abundance  of  the  dissociating 
fluid.  The  cerebral  cortex  should  be  cut  into  small  pieces  by  sec- 
tions vertical  to  the  surface.  Allow  it  to  remain  in  the  dissociator 
from  2-24  hours  ;  for  the  best  results  a  stay  in  the  fluid  of  more 
than  24  hours  is  not  so  satisfactory  ;  although  isolated  cells  are 
readily  obtained  their  processes  are  broken  off  much  nearer  the  cell 
body. 

§  ro.  Place  a  fragment  of  the  cinerea  of  the  spinal  cord  or  the 
cortex  of  the  cerebrum  on  a  clean  slide  in  a  drop  of  T^%  eosin  in 
formaldehyde  dissociator  ;  with  the  blade  of  a  scalpel  crush  the  tis- 
sue, grinding  it  thoroughly  with  a  rotary  movement,  which  will  re- 
duce it  to  small  pieces.  Gather  the  debris  together,  drain  off  the 
fluid,  and  add  a  drop  of  glycerin  containing  ^%  of  eosin.  Cover 
and  examine,  tapping  the  cover  sharply  with  the  handle  of  the 
scalpel  to  shake  out  the  processes  of  the  cells  and  free  them  from 
surrounding  matter.  Examine,  searching  for  cells  with  many  and 
long  processes.  If  a  satisfactory  preparation,  seal  according  to 

§  II0- 

§11.  Nitric  acid  dissociator.*  Formula  :  Strong  nitric  acid, 
20  c.c.  ;  water,  80  c.c.  This  fluid  is  employed  in  the  isolation  of 
muscle  fibers,  both  striated  and  plain.  The  nitric  acid  acts  upon  the 
connective  tissue  surrounding  the  muscle  fibers,  softening  and  gela- 
tinizing it  so  that  the  fibers  may  be  quite  easily  separated  from  one 
another. 

§  12.  Directions  for  use.  Place  in  the  nitric  acid  dissociator 
the  fresh  striated  muscle,  gland  or  organ  containing  the  muscle,— 
(plain  or  striated,) — that  it  is  desired  to  isolate.  If  it  is  the  inten- 
tion to  work  out  the  anatomy  of  the  muscle  or  the  relation  of  the 
muscular  coats  in  an  organ,  the  entire  muscle  or  organ  should  be 
taken  ;  otherwise,  portions  will  suffice.  At  the  ordinary  tempera- 
ture of  the  laboratory  the  dissociating  action  will  have  been  sufficient 


*  A  more  detailed  discussion  may  be  found  in  the  original  paper  :  "  Stain- 
ing and  Permanent  Preservation  of  Histological  Elements  Isolated  by  means  of 
Caustic  Potash  (KOH)  or  Nitric  Acid  (HNO3),"  by  Simon  H.  and  Mrs.  Susan- 
na Phelps  Gage.  Proc.  Am.  Soc.  of  Microscopists.  1889  :  pp.  34~45- 


86 

in  from  i  to  3  days  ;  test  at  intervals  with  needles  to  ascertain 
whether  the  fascicles  and  fibers  can  be  easily  separated  ;  or  frag- 
ments may  be  shaken  in  a  test  tube  or  vial  with  water  in  order  to 
separate  the  fibers. 

When  the  dissociation  is  sufficient  pour  off  the  acid  and  wash 
the  muscle  gently  but  thoroughly  with  water.  If  the  tissue  is  to  be 
stained  with  hematoxylin  or  carmine,  or  kept  for  any  length  of 
time,  drain  off  the  water  and  add  a  half- saturated  solution  of  alum. 
For  permanent  storage,  pour  off  the  alum  solution  and  place  suc- 
cessively in  67%  and  82%  alcohol. 

For  temporary  examination,  tease  out  a  portion  of  the  muscle  in 
water,  separating  the  fibers  carefully  by  means  of  needles  ;  cover 
and  examine. 

§  13.  Permanent  preparations,  (a}  unstained.  After  teasing 
out  with  the  needles  drain  off  the  water  and  add  a  small  drop  of 
glycerin  or  glycerin  jelly  ;  cover,  and  seal  after  first  properly  clean- 
ing (§§  no,  in).  (^)  stained.  Either  before  or  after  the  final 
teasing  stain  with  picric  alcohol,  picrofuchsin  (for  relation  of  muscle 
fiber  to  tendon),  or  after  the  alum  solution  with  hematoxylin  or  car- 
mine. Wash  away  the  staining  fluid  with  water  and  mount  (a)  in 
glycerin  or  glycerin  jelly  (§  104,  105),  or  (V)  dehydrate,  clear,  and 
mount  in  balsam  (§  107  +  ). 

§14.  Caustic  potash  dissociator.*  Formula:  Caustic  pot- 
ash, potassium  hydroxid  (in  sticks),  35—40  grams  ;  distilled  water, 
65  or  60  c.c.  This  solution  will  be  used  for  the  isolation  of  cardiac 
muscle  cells,  although  it  may  be  used  for  striated  or  plain  muscular 
tissue,  or  as  a  general  dissociator.  It  may  also  be  employed  for  iso- 
lating the  cells  of  hair,  horn  or  nail,  either  full  strength  or  diluted. 

§15.  Directions  for  use.  Place  in  the  fluid  small  pieces  of  the 
heart  muscle  of  a  fetal,  new-born  or  young  animal  ;  after  10  or  15 
minutes,  the  tissue  should  be  tested  with  needles  at  intervals  of 
about  five  minutes,  so  that  the  action  may  not  be  too  prolonged  ; 
probably  15-30  minutes  will  suffice.  As  soon  as  the  elements  sepa- 
rate readily,  pour  off  the  caustic  potash  solution  and  add  an  abund- 
ance of  60%  solution  of  potassium  acetate  (potassium  acetate,  60 
grams  ;  distilled  water,  40  c.c.).  Take  small  fragments  and  tease 
them  in  this  solution,  or  shake  them  in  a  vial,  until  the  cells  are  sep- 
arated from  each  other. 


87 

For  temporary  examination,  cover  in  a  drop  of  the  potassium 
acetate  solution.  For  permanent  preparations,  drain  off  the  potassi- 
um acetate  solution  and  add  a  small  drop  of  glycerin  or  glycerin- 
jelly. 

Stained  preparations.  Pour  off  the  potassium  acetate  solution 
and  add  a  half  saturated  solution  of  alum,  letting  it  remain  for  24 
hours  or  longer.  Tease  in  water,  stain  with  hematoxylin  or  carmine, 
wash  away  the  stain  with  water,  and  add  a  drop  of  glycerin  or  gly- 
cerin-jelly. Cover  and  seal  (§  no). 

If  a  large  amount  is  desired,  the  tissue  may  be  carried  through 
the  various  steps  in  a  vial. 

§  16.  Ranvier's  one-third  alcohol.  Formula  :  95%  alco- 
hol, t  part,  water  2  parts.  This  is  an  excellent  dissociating  fluid 
for  epithelia.  An  action  of  24  hours  is  generally  sufficient.  A 
weaker  solution  is  often  advantageous. 

§  17.  Osmic  acid.  A  -fa%  solution  of  osmic  acid  is  a  valua- 
ble dissociator,  especially  serviceable  in  the  isolation  of  nerve-fibers, 
myelinic  and  amyelinic,  and  when  fat  is  present,  since  fat  and  the 
myelin  of  myelinic  nerve-fibers  are  blackened  by  it.  12-24  hours 
generally  affords  sufficient  time  for  it  to  act. 

FIXATION. 

§  1 8.  lyiving  tissue,  when  allowed  to  die  and  remain  undis- 
turbed, gradually  loses  the  structural  features  it  had,  in  life  and 
undergoes  disintegration  and  decay.  A  fixer  is  a  fluid  (or  gas)  into 
which  the  living,  or  at  least  very  fresh,  tissue  is  placed  in  order  to 
preserve  the  structure  of  its  elements  as  nearly  as  possible  as  in  life. 

The  action  of  the  fixer  is  a  chemical  action  in  which  the  living 
substance  is  coagulated  (fixed)  by  the  reagent,  which  in  some  cases 
forms  a  chemical  union  with  the  organic  substance.  The  chemical 
action  is  generally  attended  with  a  more  or  less  marked  mechanical 
distortion,  as  shrinking  or  swelling,  to  obviate  which  chemicals  of 
opposite  tendencies  are,  in  the  best  fixers,  combined  with  each  other 
in  proportions  intended  to  neutralize  such  effects.  The  chemicals 
of  most  service  as  fixers  are  (i)  osmic  acid,  (2)  platinic  chlorid,  (3) 
picric  acid,  (4)  acetic  acid,  (5)  chromic  acid,  (6)  mercuric  chlorid,  (7) 
nitric  acid,  (8)  ethyl  (or  methyl)  alcohol  ;  also,  (9)  potassium 
dichromate,  (10)  sodium  sulphate,  (n)  copper  sulphate.  The  last 


three  are  not  as  strictly  fixers  as  hardeners.  The  following  solu- 
tions containing  these  salts,  combined  or  uncombined,  are  used  as 
fixers  in  this  course  :  (i)  Osmic  acid,  aqueous  solution  ;  (2)  Her- 
mann's fluid  ;  (3)  Flemming's  fluid  (stronger  formula)  ;  (4)  Picric 
alcohol  ;  (5)  Mercuric  chlorid,  in  aqueous  solutions  ;  (6)  Picro- 
aceto-sublimate  (vom  Rath's  fluid)  ;  (7)  Zenker's  fluid  ;  (8)  Miil- 
ler's  fluid;  (9)  Brlicki's  fluid;  (10)  Potassium  dichromate  in 
aqueous  solutions;  (n)  Chrome-oxalic;  (12)  Alcohol  (absolute— 
67%  strength)  ;  (13)  Formaldedyde  ;  also,  as  embryological  fixers, 
(14)  Perenyi's  fluid  ;  (15)  Picro-sulphuric  ;  (16)  Picro-nitric  ;  (17) 
Nitric  acid. 

§  19.  The  following  general  rules  should  be  regarded  in  the 
fixation  of  tissues  and  organs  : 

1 i )  The  volume  of  fixing-fluid  used  should  be  large,  exceed- 
ing the  volume  of  the  tissue  at  least  thirty  times.     The  less  ener- 
getic the  action  of  the  fixer,   the  greater  the  amount  of  fluid  to  be 
employed.     When  the  fluid  becomes  turbid,  it  should  be  changed  to 
fresh  at  once. 

(2)  Fix  only  as  small  pieces  of  tissue  as  possible,  or  as  practi- 
cable with  the  results  in  view.     The  block  of  tissue  should  not  be 
more  than   i   c.m.  in   any  diameter,  and,  if  possible,  let  one  of  the 
diameters  be  much  shorter, — only  one- fourth  or  one-half  of  a  centi- 
meter.    This  is  desirable  for  the  rapid -and  complete  penetration  of 
the  fixer.     Of  course,  in  the  case  of  entire  organs,  it  may  not  be  pos- 
sible to  comply  with  the  conditions. 

In  addition  to  these  two  general  principles,  there  are  four  points 
to  be  carefully  considered,  upon  which  the  excellence  in  the  results 
attained  depends  ;  they  are  (a)  the  fixer  chosen,  (&)  the  time  of  fix- 
ation, (c)  the  washing  out  of  the  fixer,  and  (d)  the  subsequent  hard- 
ening in  alcohol. 

(#)  The  choice  of  a  fluid  into  which  the  tissue  is  placed 
should  be  made  dependent  on  ( i )  a  consideration  of  the  degree  of 
excellence  of  fixation  that  is  desired  or  necessary, — whether,  in  other 
words,  details  of  cell  structure  or  the  structure  of  the  tissue  in  terms 
of  cells,  be  sought  ;  (2)  the  penetrating  power  of  the  fixer  and  the 
size  of  the  piece  of  tissue  that  it  is  necessary  to  have  ;  and  (3)  the 
stain  that  is  desired  subsequently,  which  is  largely  affected  by  the 
fixer  employed.  For  example,  Hermann's  fluid  is  possibly  the  finest 


89 

fixer  known,  but  its  penetrating  power  is  weak,  and  it  gives  good 
results  only  when  very  small  pieces  of  tissue  are  taken  ;  furthermore, 
the  number  of  stains  that  give  good  results  after  its  employment  is 
limited.  On  the  other  hand,  such  fluids  as  Miiller's  fluid  or  Er- 
licki's  fluid  should  be  avoided  when  the  preservation  of  finer  nuclear 
structures  is  desired.  Within  these  limits,  the  fixer  that  will 
give  the  best  results  in  a  special  case  must  be  determined  by  experi- 
mentation, or  the  experiments  and  results  of  others  accepted  and 
their  methods  applied. 

(b)  The  time  a  fixer  is  allowed  to  act  must  be  considered  in 
connection  with  the  character  of  the  fluid  and  the  tissue.  Often  the 
exact  limitation  of  time  is  a  matter  of  secondary  importance  ;  in 
other  cases,  however,  its  disregard  affects  the  results  seriously,  and 
as  a  general  rule,  there  is  a  minimum  and  a  maximum  time  and  be- 
tween them  an  optimum  time  that  should  be  adhered  to. 

(r)  After  the  tissue  has  been  in  the  fixing  fluid  a  proper  length 
of  time,  it  is  necessary  that  it  be  washed  thoroughly  to  remove  the 
fixer  from  it.  This  should  be  done  by  means  of  water  or  alcohol  or 
both.  In  general,  fixers  containing  salts  insoluble  in  alcohol,  or  but 
slightly  soluble,  as  osmic  acid,  chromic  acid,  potassium  dichromate, 
etc. ,  should  be  thoroughly  washed  in  water.  Fixers  containing  pic- 
ric acid  should  always  be  removed  by  alcohol  ;  mercuric  chlorid  may 
be  washed  out  by  either  water  or  alcohol. 

Inadequate  washing  out  of  the  fixer  may  either  seriously  affect 
the  cutting  quality  of  the  tissue  if  it  is  to  be  subsequently  imbedded, 
the  ease  with  which  it  can  be  stained,  or  there  may  be  formed  pre- 
cipitates in  the  tissue,  giving  illusory  effects,  distortions,  or  at  least 
a  dirty  appearance  to  the  preparation.  Time  in  properly  washing 
out  a  fixer  is  always  well  spent,  as  it  is  a  matter  for  serious  attention. 

§  20.  Resume.  In  brief,  then  :  In  fixing,  take  relatively 
large  amounts  of  fluid  and  small  pieces  of  tissue,  choose  the  fixer 
well  with  a  view  to  the  tissue  and  the  results  desired,  regulate  the 
time  carefully,  and  wash  out  thoroughly. 

HARDENING. 

§21.  Each  fixer  has  also  more  or  less  of  a  hardening  action 
upon  the  tissue.  Some  fluids  spoken  of  above  as  fixers  are  preemi- 
nently hardeners,  such  as  Miiller's  fluid  and  Erlicki's  fluid  ;  while 


go 

with  others  the  hardening  action  is  a  minimum,  e.  g.,  aqueous  solu- 
tions of  picric  acid.  The  hardening  action  of  the  fixer  is  generally 
supplemented  by  the  subsequent  use  of  alcohols  of  increasing 
strengths  (50%  to  absolute),  as  well  as  in  preparation  for  the  paraffin 
and  collodion  methods  of  imbedding.  In  fact,  with  modern  meth- 
ods of  imbedding  excessive  hardening  of  the  tissue  is  not  necessary 
and  indeed  often  should  be  avoided  as  affecting  the  cutting  quality 
of  the  tissue.  Tissue  after  fixation  has  been  completed  may  be 
stored  in  82%  or  95%  alcohol,  or  (better)  imbedded  at  once. 

FIXERS. 

$  22.  Osmic  acid.  A  very  useful  as  well  as  expensive  reagent  and  some- 
what difficult  to  use.  It  is  generally  employed  as  a  fixer  in  conjunction  with 
other  reagents,  as  in  the  mixtures  below  ($$  23  and  24).  When  used  alone  as 
a  fixer  weak  solutions  are  generally  best — T$~I%-  ^  penetrates  slowly 
and  it  "over-fixes"  cells  very  easily,  obscuring  detail  and  giving  the  parts  a 
homogeneous,  glassy  appearance.  Over-fixed  cells  cannot  be  stained,  or  with 
great  difficulty.  More  or  less  blackening  of  the  protoplasm  also  occurs.  It 
will  be  used  in  this  course  chiefly  to  demonstrate  fat,  which  is  blackened  by 
it,  and  the  zymogen  granules  of  pepsin  and  trypsin,  which  it  preserves  and 
browns  slightly. 

Fix  small  (about  %  c.  c.)  pieces  of  tissue  in  i%  osmic  acid  for  6-12  hours, 
wash  well  in  water  (running  or  changed  frequently)  for  12-24  hours,  and  place 
in  67%  and  82%  alcohols.  It  is  somewhat  difficult  to  prevent  pure  osmic  acid 
of  this  strength  from  over-fixing  the  tissue,  and  cell  detail  is  generally  lost, 
though  the  form  of  cells  is  well  preserved. 

§  23.  Hermann's  fluid.  Formula:  \%  aq.  sol.  platinic  chlorid,  15  parts  ; 
2%  aq.  sol.  osmic  acid,  4  parts  ;  glacial  acetic  acid,  i  part ;  or  you  may  take 
10%  aq.  sol.  platinic  chlorid,  3  parts  ;  \%  aq.  sol.  osmic  acid,  16  parts  ;  glacial 
acetic  acid,  2  parts  ;  water,  19  parts.  This  is  generally  recognized  as  the  finest 
fixer  known,  and  it  is  also  the  most  expensive.  The  form  and  structure  of 
cells  are  well  preserved.  It  should  only  be  employed,  however,  with  very  small 
pieces  of  tissue,  and  is  to  be  used  especially  when  cell  structure  is  to  be 
studied.  Fat  and  the  myelin  of  nerve  fibers  are  stained  black. 

Fix  in  this  1-24  hours  (or  longer — days  or  weeks  are  used  by  some),  wash 
well  in  water  (running  or  frequently  changed)  2-24  hours,  and  then  place  in 
67%  and  82%  alcohols,  12-24  hours  in  each.  In  using  this  fluid,  the  smaller 
the  pieces  taken  the  better  the  fixation  will  be,  and  in  order  that  it  be  possible 
to  obtain  a  good  stain  afterwards  tissue  should  not  be  over-fixed  and  the  fixer 
should  be  thoroughly  washed  out.  If  there  is  a  blackening  of  the  tissue,  or  a 
precipitate  in  it,  both  may  be  removed  by  treatment  of  the  sections  on  the 
slide  with  a  10-20%  solution  of  hydrogen  dioxid  in  67%  alcohol.  Employ 


after  Hermann's  fluid,  as  stains,  Heidenhain's  iron  hematoxylin,  Delafield's 
hematoxylin,  safranin  (as  a  red  stain),  or  gentian  violet  (as  a  blue  stain). 

\  24.  -Flemming's  fluid  (Chrome-aceto-osmic).  Formula:  i%  aq.  sol. 
chromic  acid,  15  parts;  2%  aq.  sol.  osmic  acid,  4  parts;  glacial  acetic  acid,  i 
part;  or,  ro  aq.  sol.  chromic  acid,  3  parts  ;  i%  osmic  acid,  1 6  parts  ;  glacial 
acetic  acid  2  parts  ;  water,  19  parts.  This  is  a  fine  fixer  and  in  most  cases  gives  as 
good  results  as  Hermann's,  and  is  not  as  expensive.  It  browns  tissue  less,  and 
while  it  blackens  fat,  does  not  blacken  the  myelin  of  myelinic  nerve  fibers 
as  does  Hermann's.  It  should  be  employed  in  general  in  the  same  cases  and 
in  the  same  way  as  Hermann's  fluid. 

Fix  tissue  1-24  hours  (or  longer);  wash  well  in  running  water  2-24  hours  ; 
place  in  67%  and  82%  alcohols,  12-24  hours  in  each.  Bleaching  of  the  sections 
may  be  necessary,  as  with  Hermann's  fluid.  Take  only  very  small  pieces  of 
tissue.  Employ  the  same  stains  as  with  Hermann's  fluid. 

\  25.  Picric  alcohol.  Formula:  Distilled  water,  250  c.  c. ;  95%  alcohol, 
250  c.  c. ;  picric  acid,  i  gram.  A  delicate  fixer,  penetrates  well,  and  does  not 
make  the  tissue  brittle  or  tough.  It  may  be  used  with  most  tissues  and  organs. 

Allow  it  to  act  1-3  days,  changing  to  fresh  each  day  if  the  bulk  of  tissue 
is  large,  transfer  to  67%  alcohol  for  1-2  days,  82%  alcohol  several  days,  chang- 
ing several  times.  If  the  paraffin  method  of  imbedding  is  to  be  employed  it 
is  best  to  leave  the  tissue  in  82%  alcohol  until  no  more  or  very  little  color 
comes  away  from  it,  as  otherwise  it  affects  the  cutting  quality  of  the  tissue. 
Most  staining  methods  may  be  employed.  Staining  is  good,  though  not  quite 
as  brilliant  as  with  mercuric  chlorid  fixers. 

%  26.  Mercuric  chlorid.  One  may  employ  (a)  a  saturated  solution  in 
water,  or  better,  (b)  a  saturated  solution  in  normal  salt  solution  with  1-5%  gla- 
cial acetic  acid.  Water  will  dissolve  about  5%,  normal  salt  solution  about  12% 
of  the  mercuric  chlorid.  This  is  a  good  fixer,  especially  when  the  piece  is 
small.  It  fixes  as  soon  as  it  penetrates  and  is  apt  to  make  tissue  brittle  if  it  is 
left  too  long.  Staining  after  it  is  brilliant.  The  larger  percentage  of  acetic 
acid  is,  perhaps,  to  be  preferred  for  most  histological  objects. 

Fix  the  fresh  tissue  ^-24  hours  according  to  the  size  of  the  piece.  Re- 
move to  67%  alcohol  for  1-2  days,  82%  alcohol  several  days,  changing  often. 
The  82%  alcohol  should  contain  enough  tincture  of  iodin  to  give  it  a  yellow 
color,  and  fresh  tincture  added  or  (better)  the  alcohol  changed  when  the  yel- 
low color  of  the  iodin  in  the  alcohol  is  lost.  As  long  as  the  alcohol  is  decol- 
orized, washing  should  be  continued,  since  it  is  important  that  the  mercuric 
chlorid  be  all  removed  from  the  tissue  ;  otherwise  precipitates  will  form  in  the 
preparation  after  it  is  mounted  or  before,  and  spoil  the  result.  Wash  out  in 
alcohol  thoroughly  and  carefully.  Almost  any  stain  may  be  employed  after  a 
mercuric  chlorid  fixation. 

g  27.  Picro-aceto-sublimate.  (vom  Rath' s  fluid).  Formula :  Saturated 
aqueous  solution  of  picric  acid,  50  c.c.  ;  saturated  aqueous  solution  of  mercuric 
chlorid,  50  c.c.  ;  glacial  acetic  acid,  i  c.c.  (1-5  c.c.  according  to  the  organ). 
This  is  quite  a  satisfactory  fixer  if  care  is  taken  in  its  employment,  combining 


92 

to  a  certain  extent  the  advantages  of  picric  acid  and  mercuric  chlorid.  A 
stronger  percentage  of  acetic  acid  (4  or  5%)  is  to  be  preferred  with  organs  con- 
taining a  large  amount  of  connective  tissue. 

Fix  the  object  for  1-24  hours  ;  place  in  67%  alcohol  for  1-2  days,  82%  alco- 
hol for  several  days,  changing  several  times,  whenever  the  alcohol  has  become 
colored  yellow.  The  tissue  should  be  left  in  the  82%  alcohol  until  almost  all 
the  picric  acid  has  been  washed  out  and  the  alcohol  no  longer  becomes  colored, 
or  but  slightly.  Tincture  of  iodin  may  be  used  as  with  mercuric  chlorid 
tissue,  but  is  not  necessary.  Stain  as  you  like. 

$28.  Zenker's  fluid.  Formula:  Potassium  dichromate,  2.5  gr. ;  sodium 
sulphate,  i  gram;  mercuric  chlorid,  5  gr. ;  water,  100  c.  c.;  and  add  before 
using,  glacial  acetic  acid,  5  c.  c.  This  is  a  fluid  used  quite  generally  of  late. 
The  potassium  dichromate  seems  to  check  the  brittleness  that  the  mercuric 
chlorid  would  cause,  otherwise  its  advantages  and  faults  are  those  of  mercuric 
chlorid  ;  staining  after  it,  however,  is  apt  to  be  a  little  more  difficult  and  not 
as  brilliant  as  with  mercuric  chlorid  fixation.  Its  penetration  is  surprisingly 
good. 

Fix  in  Zenker's  fluid  12-48  hours,  wash  well  in  water,  running  or  fre- 
quently changed,  12-48  hours,  to  remove  the  dichromate  ;  transfer  to  67%  alco- 
hol for  1-2  days,  82%  alcohol  for  several  days,  keeping  in  the  dark  while  in 
the  alcohol.  To  the  82%  alcohol  add  a  drop  or  so  of  tincture  of  iodin,  adding 
fresh  iodin  or  changing  the  alcohol  when  the  color  is  lost.  This  should  be 
continued  as  long  as  the  iodinized  alcohol  is  decolorized  in  order  to  avoid  a 
precipitate  of  the  mercuric  chlorid  in  the  tissue.  Avoid,  however,  adding  an 
excess  of  iodin, -since  it  will  affect  the  s  taining  quality  of  the  tissue.  Stain  as 
you  wish. 

$  29.  Miiller's  fluid.  Formula  :  Potassiurii  dichromate,  2.5  grams;  sodium 
sulphate,  i  gram  ;  water,  100  c.c.  This  fluid  is  more  of  a  hardener  than  a  fixer  ; 
it  should  be  avoided  (as  likewise  Erlicki's  fluid  and  potassium  dichromate) 
when  the  preservation  of  nuclear  structure  is  desired.  Staining  after  its  use  is 
sometimes  difficult.  It  is,  however,  useful  for  general  work. 

Place  the  object  in  an  abundance  of  the  fluid  and  harden  for  from  1-8 
weeks,  changing  the  fluid  at  first  each  day.  In  general,  10-14  days  will  be  suf- 
ficient. Wash  in  running  water  for  24-48  hours  or  longer,  remove  to  67%  alco- 
hol for  1-2  days,  82%  alcohol  several  days.  Keep  in  the  dark  while  in  the  alco- 
hols, and  change  to  fresh  when  the  fluid  is  colored  yellow.  Tissue  hardened 
in  Miiller's  fluid  cuts  well,  and  it  is  useful  in  preparing  sections  of  large  organs, 
or  organs  with  much  connective  tissue. 

%  30.  Erlicki's  fluid.  Formula  :  Potassum  dichromate,  2.5  grams  ;  copper 
sulphate,  i  gram  ;  water,  100  c.c.  This  is  quite  similar  to  Miiller's  fluid  in  its 
action  and  results,  save  that  its  action  is  more  rapid  and  stronger.  Therefore, 
it  had  better  be  employed  with  smaller  objects,  and  allowed  to  act  only  2-14 
days.  Otherwise,  employ  like  Miiller's  fluid. 

\  31.     Potassium  dichromate.     2%,  3%,  and  5%  aqueous  solutions. 


93 

This  is  quite  similar  to  M  tiller's  fluid  in  its  action,  and  may  be  employed  in  the 
same  cases.  It  is  generally  used  for  the  central  nervous  system. 

Harden  in  an  abundance  of  the  solution  for  2-8  weeks,  beginning  with  the 
2%  solution  for  2-6  days,  3%  solution  1-3  weeks,  5%  solution  1-3  weeks. 
Wash  out  in  running  water  24-48  hours.  Place  in  67%  and  82%  alcohols  sev- 
eral days,  keeping  in  the  dark  meanwhile,  changing  when  the  alcohol  is 
colored. 

I  32.  Chrome-oxalic  (Graf).  Formula:  8%  aqueous  solution  of  oxalic 
acid,  4  parts  ;  95%  alcohol,  3  parts  ;  i%  chromic  acid,  3  parts.  Mix  the  solu- 
tions in  the  order  given. 

Fix  tissue  in  the  solution  1-6  hours,  wash  in  running  water  >£-i  hour,  and 
harden  in  50%,  67%,  and  82%  alcohols. 

This  appears  to  be  a  good  general  fixer  for  many  organs  ;  it  penetrates 
well,  the  tissue  cuts  well  after  this  method  of  fixation,  and  the  staining  is  sharp 
and  strong. 

%  33.  Alcohol.  95%,  67-70%,  absolute  alcohol.  The  employment 

of  most  of  the  fixers  so  far  mentioned  requires  the  expenditure  of  considerable 
time,  rendering  them  inapplicable  or  unsuitable  in  many  instances.  95%  alco- 
hol itself  answers  admirably  for  most  histologic  objects,  fixing  well,  hardening 
and  likewise  dehydrating  (\  41)  preparatory  to  imbedding  in  paraffin  or  col- 
lodion, affording  thus  a  considerable  economy  of  time.  It  is  also  most  service- 
able in  pathological  tissues  where  the  presence  of  bacteria  is  suspected.  In 
some  instances  67%  alcohol  answers  as  well  or  better,  while  in  other  cases  ab- 
solute (99%)  alcohol  should  be  employed.  The  addition  of  5%  glacial  acetic 
acid  increases  the  penetrating  power  and  improves  the  cutting  quality  of  objects 
containing  much  connective  tissue. 

Fix  in  95%  alcohol  for  1-3  days,  changing  two  or  three  times,  after  3  or  4 
and  after  24  hours.  The  tissue  will  probably  be  found  to  be  dehydrated  and 
ready  for  the  next  step  of  the  imbedding  process  ($  44  or  52).  Stain  as  you 
wish. 

\  34.  Formaldehyde.  Solutions  of  this  chemical  have  been  found  to  be 
good  preservatives  and  hardeners  and  fairly  good  fixers.  It  penetrates  rapidly, 
and  preserves -the  natural  transparency  and  pigmentation  of  the  tissue,  making 
it  valuable  for  gross  anatomy  and  museum  purposes.  As  a  fixer,  an  aqueous 
solution  of  2-4%  strength  may  be  employed,  or  it  may  be  used  in  conjunc- 
tion with  other  chemicals,  as  picric  acid,  inpicro-formalin,  or  chromic  acid  and 
acetic  acid. 

Fix  12-24  hours,  remove  to  67%  alcohol  for  a  day,  82%  alcohol  one  to 
several  days.  Stain  as  you  wish. 

$  35.  Perenyi's  fluid.  Formula  :  10%  aq.  sol.  nitric  acid,  4  parts  ;  95% 
alcohol,  3  parts  ;  >£%  aq.  sol.  chromic  acid,  3  'parts.  An  embryological  fixer 
of  much  value.  It  is  also  serviceable  for  general  work. 

Fix  tissue  for  4-5  hours,  place  in  67%  alcohol  for  24  hours,  82%  alcohol 
several  days. 

|  36.     Picro-nitric.     Formula:  Water,  95  c.c.  ;  nitric  acid,  5  c.c.  ;  picric 


94 

acid,  to  saturation.  This  is  a  valuable  embryological  fixer,  especially  service- 
able with  eggs  that  have  a  good  deal  of  yolk. 

Fix  for  4-12  hours,  transfer  to  67%  alcohol  for  i  day,  82%  alcohol,  several 
days.  It  is  necessary  to  wash  the  tissue  well  in  several  changes  of  the  alcohol, 
as  the  picro-nitric  is  washed  out  with  difficulty. 

|  37.  Picro-sulphuric.  Formula :  Distilled  water,  100  c.c.  ;  sulphuric 
acid,  2  c.c.  ;  picric  acid,  to  saturation. 

This  may  be  used  full  strength  or  diluted  with  1-3  volumes  of  distilled 
water.  It  is  a  embryological  fixer,  useful  for  many  eggs,  chiefly  invertebrate, 
and  for  many  invertebrate  animals.  In  general,  fix  for  1-6  hours,  remove  to 
€>7%  (7°%)  alcohol  for  a  day,  and  place  in  82%  alcohol,  in  which  it  should 
remain  until  most  or  all  of  the  yellow  color  has  been  extracted. 

$  38.  Nitric  acid.  A  10%  solution  of  nitric  acid  is  serviceable  in  fixing 
the  blastoderm  of  the  chick. 

SECTIONING. 


§  39.  In  addition  to  the  examination  of  tissue  by  the  separa- 
tion of  the  component  elements — isolation— it  may  be  examined 
microscopically  after  cutting  very  thin  slices  or  sections  of  it.  This 
may  be  done  free-hand  or  by  means  of  a  special  machine,  a  micro- 
tome, and  with  or  without  an  imbedding  and  supporting  mass. 

For  the  finer  work  an  imbedding  mass  and  a  microtome  must 
be  used.  Free-hand  sectioning  without  an  imbedding  mass,  and 
even  without  previous  hardening,  is,  however,  necessary  or  advis- 
able when  economy  of  time  is  a  desideratum,  as  in  clinical  exam- 
inations of  tissue,  when  one  wishes  to  study  the  part  alive  or  fresh 
(i.  e.,  not  treated  with  reagents),  or  if  the  reagents  necessary  for 
fixing  and  imbedding  destroy  or  alter  the  structural  features  to  be 
investigated. 

The  ability  to  recognize  tissues  and  organs  unaffected  by  re- 
agents and  without  the  employment  of  methods  involving  the  ex- 
penditure of  time  and  effort  is  very  desirable,  especially  in  patho- 
logical work,  when  haste  often  forbids  the  employment  of  the  finer 
methods,  were  facilities  for  their  use  available,  as  in  many  cases 
they  are  not.  Great  skill  in  the  use  of  simple  tools  may  be  gained 
and  counts  for  much.  It  should  be  remembered  also  that  the 
greater  one's  knowledge  of  a  structure  the  less  the  need  to  resort  to 
special  methods  of  preparation  for  its  recognition. 


95 
IMBEDDING  METHODS. 

§  40.  When  the  consideration  of  time  is  not  so  important  and 
finer  results  are  more  to  be  desired,  the  sections  should  be  prepared 
according  to  some  method  in  which  an  imbedding  mass  is  used. 
The  interstices  of  the  tissue  are  completely  filled  with  some  sub- 
stance that  will  give  support  and  greater  consistency  and  homegen- 
eity  to  the  tissue,  and  thereby  enable  the  cutting  of  much  thinner 
and  more  perfect  sections. 

There  are  three  methods  that  are  generally  employed,  (#)  the 
Paraffin  method,  (£)  the  Collodion  method,  and  (c)  the  Freezing 
method ;  the  imbedding  masses  to  fill  the  spaces  being  respectively 
paraffin,  collodion  and  a  congelation  mass, — ice.  The  last  is  the 
simplest  ;  it  requires  less  expenditure  of  time,  fewer  reagents,  and 
its  results  are  the  crudest.  As  in  cutting  free-hand  sections  without 
imbedding,  the  freezing  microtome  should  be  employed  when  haste 
is  necessary  and  finer  detail  unimportant,  as  in  clinical  work.  The 
two  remaining  methods  may  be  employed  in  most  cases  and  give 
good  results.  A  choice  between  them  must  be  determined  by  the 
following  considerations  and  the  special  exigencies  of  the  case  : 

(i)  As  a  general  rule  paraffin  may  be  employed  when  very 
thin  sections  are  desired  and  the  piece  or  organ  is  not  very  large  ; 
collodion,  when  larger  sections  are  necessary  but  which  need  not  be 
so  thin.  (2)  For  paraffin,  heat  is  necessary,  which  is  not  required 
in  the  collodion  method.  (3)  In  the  case  of  paraffin,  the  imbedding 
mass  is  removed  from  the  sections  before  they  are  stained  and 
mounted  ;  the  collodion  is  (usually)  not  dissolved  out.  With  most 
nuclear  stains  the  collodion  is  colored  more  or  less,  affecting  the  ap- 
pearance and  excellence  of  the  preparation  unless  it  is  bleached,  a 
process  not  possible  in  many  cases.  (4)  In  paraffin  sections  there 
is  apt  to  be  more  or  less  crushing  together  of  the  parts  of  the  tissue  ; 
by  the  employment  of  collodion,  the  form  of  the  organ  may  be  more 
exactly  preserved,  sometimes  an  important  consideration. 

§  41.  The  following  will  indicate  the  steps  in  the  employment 
of  the  two  methods  : 


96 
Living  tissue 

v 

Fixing 
Hardening  in 
alcohols  (50-67-82-95  %) 


Dehydration 
(in  95-99  %  alcohol) 


Paraffin  Method 


Cedar-oil Clearing . 


Melted  paraffin Infiltrating < 


Collodion  Method 


Ether-alcohol 


Thin  collodion 
(1-2  %  solutions) 

V 

Thick  collodion 
(6-8  %  solutions) 


Paraffin  mass  4 

cooled  quickly Imbedding 


(  .  v 

I  Collodion  hardened 
in  chloroform 

\         I 


Collodion  block 
clarified 


In  Paraffin  ---------  Sectioning  ----------  In  Collodion 


THE   PARAFFIN    METHOD. 


§  42.  As  seen  by  the  above  scheme,  the  aim  is  to  fill  all  the 
interstices  of  the  tissue  with  paraffin  of  the  right  degree  of  hardness 
to  have  it  cut  well.  Paraffin  is  not  soluble  in  water  or  alcohol,  but 
is  soluble  in  a  number  of  fluids  which  in  turn  are  miscible  with  alco- 
hol. Hence  the  following  steps  are  necessary  :  (i)  the  tissue  must 
be  first  water-rid,  thoroughly  dehydrated  with  strong  alcohol;  (2) 
freed  from  the  alcohol,  cleared  by  a  fluid  that  mixes  with  melted 
paraffin,  which  (3)  takes  the  place  of  the  clearer  in  the  tissue, 
infiltrates  it,  filling  the  spaces  ;  (4)  finally,  the  tissue  is  imbedded  in 
paraffin  of  the  right  degree  of  hardness,  the  mass  cooled,  and  it  is 
ready  (5)  to  cut,  or  section. 


97 

§43-  Dehydration.  After  the  various  steps  pertaining  to  the 
proper  fixing  and  hardening  (§21)  of  the  tissue  have  been  prop- 
erly pursued  it  may  be  stored  in  alcohol  of  82^-95%  strength  de- 
pending on  the  tissue  and  its  purpose.  The  dehydration  necessary 
in  imbedding  may  be  accomplished  by  immersion  in  alcohol  of  95% 
strength.  For  more  delicate  work  it  is  perhaps  better  to  employ 
stronger  (absolute)  alcohol. 

Immerse  small  pieces  2-3  mm.  in  diameter  for  at  least  6-8 
hours  in  95%  alcohol  changed  once  or  twice.  A  longer  time,  even 
days,  does  no  harm  and  is  preferable  to  ensure  complete  dehydra- 
tion. For  larger  pieces  of  tissue  or  entire  organs  a  correspondingly 
longer  period  of  dehydration  should  be  employed,  a  several  days' 
stay,  with  the  alcohol  changed  daily,  being  often  advisable,  In  any 
case,  dehydrate  thoroughly,  changing  the  alcohol  1-3  times,  letting 
the  tissue  remain  in  it  for  a  longer  rather  than  a  shorter  period. 

§  44.  Clearing.  The  alcohol  must  next  be  replaced  by  some 
solvent  of  paraffin  which  is  miscible  with  alcohol, — a  step  spoken  of 
as  clearing .  Cedar-wood  oil  is  perhaps  most  generally  serviceable, 
although  for  special  purposes  other  media,  such  as  xylol,  bergamot 
oil,  or  chloroform  may  be  preferred.  When  the  tissue  is  dehydrat- 
ed, it  is  removed  to  a  vial  of  thickened  cedar- wood  oil.  It  will  float 
at  first,  but  as  the  alcohol  used  for  dehydration  is  displaced  by  the 
oil,  it  sinks  to  the  bottom,  the  currents  of  alcohol  rising  from  it. 
When  the  alcohol  is  entirely  removed  by  the  oil,  such  currents  will 
have  ceased  to  rise  from  it  and  the  tissue  will  be  clear  and  translu- 
cent,— except,  of  course,  such  as  is  dark  in  color. 

§  45.  Infiltration.  •  After  the  tissue  is  completely  cleared  by 
the  cedar- wood  oil,  remove  it  to  a  dish  of  melted  infiltration  paraffin 
and  place  in  the  paraffin  oven  for  2  to  24  hours,  depending  on 
the  size  of  the  piece.  Quite  large  pieces  may  be  left  longer.  The 
melted  paraffin  replaces  the  cedar-wood  oil,  filling  in  the  interstices 
of  the  tissue.  Paraffin  melting  at  about  48°  C.  is.used,  and  is  pre- 
pared by  mixing  equal  parts  of  43°  and  54°. paraffin.  It  is  best  not  to 
expose  to  a  higher  temperature  than  is  necessary,  or  for  a  long  period 
of  time.  The  paraffin  oven  will  be  maintained  at  a  temperature  of 
5o°-52°  C. 

§  46.  Imbedding.  It  is  best  to  use  fresh  paraffin  for  imbed- 
ding and  generally  with  a  melting  point  higher  than  that  of  the  infil- 


98 

tration  paraffin, — 50°  C.  paraffin  (42°  paraffin  i  part,  54°  paraffin  2 
parts),  answers  well  in  a  room  of  19°  to  20°  C.,  and  will  be  gener- 
ally used.  If  the  cutting  is  to  be  done  in  a  room  of  lower  tempera- 
ture, a  softer  grade  of  paraffin  may  be  used  for  imbedding  ;  if  at  a 
higher  temperature,  a  harder  paraffin  should  be  chosen  ;  54°  C.  par- 
affin giving  good  results  when  summer  work  is  necessary. 

Make  a  small  paper  box,  fill  it  with  the  melted  imbedding 
paraffin  ;  transfer  to  it  the  tissue  from  the  paraffin  oven,  arrange  it 
carefully  in  the  box  in  the  way  you  wish  it  for  cutting,  and  cool  the 
mass  by  floating  the  box  on  a  dish  of  cold  water. 

§  47.  In  imbedding  in  paraffin  observe  the  following  rules  ; 
(i)  Take  no  more  paraffin  (no  larger  box)  than  is  needed  to  form  a 
mass  of  convenient  size  around  the  specimen.  The  aim  is  to  have 
as  homogeneous  a  mass  as  possible  ;  paraffin  tends  to  crystallize  if 
it  cools  slowly,  hence  the  smaller  the  mass  the  more  rapidly  may  it 
be  cooled.  (2)  Let  the  imbedding  paraffin  when  poured  into  the 
box  be  several  degrees  above  its  melting  point,  and  the  tissue  like- 
wise should  have  an  equal  temperature.  Should  the  imbedding 
paraffin  (or  the  tissue)  be  too  cool  it  will  not  set  well  around  the 
specimen,  and  a  film  of  air  may  be  enclosed.  On  the  other  hand, 
take  care  that  the  paraffin  is  not  hot  enough  to  "cook"  the  tissue, 
thereby  shrinking  it  and  rendering  it  hard  and  tough  or  ruining  it 
altogether.  (3)  Cool  by  floating  on  cold  water.  Paraffin  in  cooling 
must  contract  greatly  if  it  does  not  crystallize  ;  the  more  homo- 
geneous it  is  the  more  it  must  contract,  and  if  it  is  cooled  on  all 
sides  it  will  crystallize  in  the  center  of  the  mass,  because  it  cannot 
shrink. 

§  48.  Cutting  the  sections.  The  essentials  for  good  paraffin 
sectioning  are  (i)  well-imbedded  tissue,  (2)  a  sharp  knife  (or  sec- 
tion razor),  (3)  a  room  of  the  proper  temperature,  and  (4)  the  par- 
affin block  properly  trimmed  and  arranged  in  the  microtome.  Fur- 
thermore, tissue  fixed  and  hardened  in  different  ways  cuts  very  dif- 
ferently. Tissue  fixed  in  Hermann's,  Flemming's,  Miiller's,  or 
Zenker's  fluid  cuts  well  ;  picric  acid  and  mercuric  chlorid  tissue  is 
more  apt  to  be  tough  or  hard,  etc.  The  different  organs  and  tissues 
have  of  course  very  different  adaptabilities  to  the  method. 

After  the  imbedding  mass  is  well  cooled,  remove  the  paper  box 
and  trim  the  part  containing  the  tissue  in  a  pyramidal  form,  two  of 


99 

the  sides  at  least  being  as  nearly  parallel  as  possible.  Clamp  the 
block  of  paraffin  in  the  holder  of  the  microtome  so  that  the  tissue 
will  be  at  the  proper  level  for  cutting,  being  careful  to  have  the  par- 
allel sides  also  parallel  to  the  edge  of  the  knife.  If  a  ribbon  micro- 
tome is  used,  heat  the  holder  and  melt  the  end  of  the  block  upon  it. 
Cool  and  place  the  holder  in  its  place  in  the  microtome,  again  hav- 
ing the  parallel  sides  and  the  edge  of  the  knife  parallel.  Use  a  very 
sharp,  dry  razor  for  cutting  the  sections.  Clamp  it  in  the  (Minot) 
microtome  slightly  inclined  to  the  cutting  surface  of  the  tissue.  If 
the  temperature  of  the  room  is  right  for  the  paraffin  used,  the  sec- 
tions will  remain  flat,  and  if  the  directions  given  above  for  trimming 
and  arranging  the  block  be  observed,  they  will  adhere  and  thus  form 
a  ribbon.  If  the  room  is  too  cold  or  the  paraffin  too  hard,  the  sec- 
tions will  roll  ;  if  it  is  too  warm,  the  sections  will  crush  or  be  imper- 
fect. If  a  microtome  in  which  the  knife  is  not  fixed,  is  used,  make 
the  sections  with  a  rapid  straight  cut  as  in  planing.  Do  not  try  to 
section  with  a  drawing  cut  as  used  in  collodion  sectioning.  io//  will 
be  found  the  most  convenient  thickness  for  the  sections,  though  in 
special  cases  they  should  be  thinner  or  even  thicker. 

Remember  to  have  the  paraffin  block  trimmed  with  two  sides 
parallel  and  the  knife  edge  parallel  to  these.  Also,  do  not  attempt 
to  cut  if  the  temperature  of  the  room  is  too  high, — above  21°  C. 

§  49.  Resume  of  the  method.  To  obtain  as  good  results  as 
possible  with  a  certain  organ  fixed  and  hardened  in  a  certain  way, 
the  steps  must  be  carefully  and  exactly  followed.  Let  the  dehydra- 
tion be  complete,  clearing  thorough,  infiltration  sufficient  ;  imbed, 
carefully  observing  the  three  cautions  mentioned  ;  and  in  cutting, 
remember  to  have  a  sharp  knife,  a  cool  room,  and  the  imbedding 
block  properly  trimmed.  Success  also  depends  largely  on  the  pre- 
vious treatment  in  the  fixer  and  on  the  care  with  which  the  fixer  is 
washed  out. 

Properly  employed,  the  paraffin  method  is  widely  serviceable, 
being  only  useless  where  the  tissue  is  very  large,  very  hard,  hard- 
ened or  injured  by  heat,  or  where  the  exact  form  of  a  large  organ  is 
important. 

THE   COIvLODION   METHOD. 

§  50.  A  comparison  with  the  paraffin  method  has  already  been 
given  (§40);  there  may  be  emphasized  here  three  points:  (i) 


100 

with  paraffin  heat  is  required,  with  collodion  no  heat  ;  (2)  paraffin 
must  be  removed  from  the  sections  subsequently,  collodion  need  not 
and  usually  is  not  dissolved  out  ;  (3)  by  the  paraffin  method  may 
be  obtained  small  sections  (2  cm.  square  or  less),  and  thin,  by  the 
collodion,  larger  sections,  but  thicker.  With  paraffin  heat  (melting 
and  cooling)  is  necessary,  and  the  mass  is  sometimes  spoken  of  as  a 
fusion  imbedding  mass  ;  collodion  is  a  solution,  arid  the  mass  is  left 
in  the  tissues  by  evaporation  or  its  equivalent. 

In  the  collodion  method  the  imbedding  mass  with  which  the 
spaces  of  the  tissue  are  to  be  filled  is  collodion,  a  solution  of  pyroxy- 
lin (soluble  cotton)  in  ether  and  alcohol,  hence  the  steps,  which  are 
comparable  with  those  of  the  paraffin  method  (see  §  41),  are  (i) 
Dehydration,  removal  of  the  water  ;  (2)  Saturation  with  ether-alco- 
hol, the  solvent  of  the  collodion  ;  (3)  Infiltration  with  collodion 
solutions,  a  thin  and  a  thick  ;  (4)  Imbedding  in  a  thick  collodion 
mass,  which  is  hardened  and  (as  employed  in  this  course)  clarified 
and  (5)  sections  cut. 

.§51.  Dehydration.  Let  it  be  complete,  as  in  the  prepara- 
tion for  paraffin  imbedding  (§  43).  Immerse  the  tissue  in  95% 
alcohol  for  12-24  hours  or  longer,  changing  1-3  times.  Consult  also 
§  43  upon  the  dehydration  of  tissue. 

§  52.  Saturation  with  ether-alcohol  (equal  parts).  Re- 
move the  tissue  from  the  strong  alcohol  and  place  it  in  a  vial  of 
ether-alcohol  for  12-24  hours.  In  addition  to  preparing  the  tissue 
for  the  collodion  solutions,  it  completes  the  dehydration,  should  it 
be  imperfect.  In  special  cases,  or  if  the  dehydration  is  very 
thorough  and  the  specimen  small,  this  step  may  be  omitted.  A 
satisfactory  infiltration  is,  however,  more  certain  if  ether-alcohol 
be  used. 

§53.  Infiltration:  (a)  with  thin  collodion.  Pour  off  the 
ether-alcohol  and  add  the  thin  (i^%)  solution  of  collodion  (§  154). 
This,  being  a  solution  in  ether-alcohol  with  which  the  tissue  is  sat- 
urated, readily  permeates  it.  It  is  best  to  allow  at  least  a  day  for 
this  to  take  place,  although  if  there  is  time  a  stay  of  several  days  is 
better,  there  being  little  or  no  danger  of  deterioration  while  in  the 
solution.  With  large  (i  c.  c.  +  )  objects  an  infiltration  of  a  week  or 
even  a  month  is  advisable. 

§  54.      Infiltration  :   (b)  with  thick  collodion.     Pour  off  the 


101 

thin  collodion  solution  and  add  thick  (6%)  solution.  In  this  there  is 
gradual  concentration  of  the  solution  in  the  tissue.  Allow  small 
specimens  to  remain  a  day,  or,  better,  several  days  ;  larger  objects 
should  be  given  a  proportionately  longer  time,  a  week  to  a  month, 
or  even  longer. 

If  the  object  to  be  imbedded,  such  as  many  embryological 
specimens,  is  one  with  large  interior  cavities  with  thin  walls  the 
transfer  from  the  thin  solution  to  the  thick  solution  may  be  attended 
by  a  collapse  of  the  walls  and  a  consequent  shriveling  and  distortion 
of  the  specimen.  Avoid  this  by  allowing  the  thin  solution  to 
thicken  very  gradually  by  evaporation  until  the  solution  has  at- 
tained the  right  consistency.  To  accomplish  this  it  is  only  neces- 
sary to  have  the  cork  of  the  vial  containing  the  specimen  perforated 
by  a  small  hole.  A  small  piece  of  paper  may  be  inserted  with  the 
cork,  or  with  porous  corks  no  special  effort  need  be  made.  Unless 
the  thick  solution  has  itself  thickened  by  evaporation,  with  large 
specimens  it  is  advisable  to  follow  the  6%  bath  with  a  stay  in  a 
thicker  solution,  as  8%,  for  a  day  or  so. 

§  55.  Imbedding.  Pour  off  the  6%  solution  and  add  for  a 
short  time  at  least  an  8%  solution  of  collodion.  The  tissue  is  now 
ready  for  imbedding  in  8%,  which  may  be  accomplished  in  either  of 
two  ways  :  (a)  on  a  cork  or  other  holder  that  may  be  clamped  in  the 
microtome,  or  (b)  in  a  paper  box.  Only  those  specimens  need  be 
imbedded  in  a  box  that,  from  their  shape,  or  for  purposes  of  careful 
orientation  or  serial  sectioning,  require  a  larger  imbedding  mass 
around  them. 

(a)  On  a  holder  (cork).  Choose  a  cork  of  a  convenient  size  ; 
put  a  drop  or  two  of  collodion  upon  one  end  and  insert  a  pin  verti- 
cally to  the  surface  near  the  edge.  Transfer  the  tissue  from  the 
vial  of  thick  collodion  to  the  cork  and  lean  it  against  the  pin.  The 
shape  of  many  tissues  will  obviate  the  need  of  a  pin.  Pour  the 
thick  collodion  onto  the  tissue,  drop  by  drop,  moving  the  cork  in 
such  a  way  that  the  thick  viscid  mass  may  be  made  to  surround 
and  envelop  the  tissue.  Continue  to  add  drops  of  collodion  at  in- 
tervals until  the  tissue  is  well  surrounded,  and  then  as  soon  as  a 
slight  film  hardens  on  the  surface  invert  the  cork  bearing  the  tissue 
in  a  shell-vial  of  large  diameter  containing  enough  chloroform  to 
float  the  specimen  and  cork.  If  the  piece  of  tissue  is  of  awkward 


102 

size  and  shape,  oiled  paper  may  be  wound  around  the  end  of  the 
cork  and  tightly  tied,  the  projecting  hollow  cylinder  being  long 
enough  to  receive  the  object.  The  tissue  may  be  put  into  the  cyl- 
inder as  before,  the  collodion  slowly  poured  in  drop  by  drop  until 
the  specimen  is  completely  covered.  When  a  film  has  formed, 
place  in  chloroform  as  before. 

(b).  In  a  paper  box.  When  a  box  is  required  for  imbedding 
proceed  as  follows  :  The  inside  of  the  paper  box  should  be  slightly 
oily  to  prevent  the  collodion  from  sticking  to  it.  Rub  upon  the 
paper  that  is  to  be  folded  to  form  the  box  a  little  vaseline,  and  then 
with  a  cloth  or  lens  paper  remove  as  much  as  possible.  Fold  the 
paper  into  a  box  of  convenient  size  and  shape.  Remove  the  object 
from  the  thick  collodion  and  place  it  in  the  box,  arranging  it  in  the 
manner  wished  with  a  view  to  sectioning  it  later.  Pour  over  it 
slowly,  drop  by  drop  or  a  little  at  a  time,  an  8%  solution  of  collodion 
until  the  specimen  is  well  covered  and  the  box  sufficicently  filled. 
It  is  better  to  have  a  deep  layer  over  the  specimen.  The  8%  solu- 
tion does  not  afford  the  best  mass  for  cutting,  so  that,  with  large 
objects,  it  is  better  to  allow  the  mass  in  the  box  to  thicken  by  evap- 
orating it  slowly  under  a  bell-jar  (aquarium  jar)  until  it  has  at- 
tained the  consistency  of  a  very  thick  syrup.  When  thick  enough 
allow  a  film  to  harden  upon  the  surface  and  immerse  it  in  a  glass 
box  or  jar  of  chloroform. 

§  56.  Hardening  and  clarifying.  The  chloroform  into 
which  the  collodion  mass  is  placed  takes  out  the  ether- alcohol  and 
hardens  the  collodion  mass,  for  which  a  few  hours  is  sufficient. 
Allow  the  chloroform  to  act  for  6-24  hours.  If  it  acts  long  enough 
the  imbedding  mass  is  rendered  entirely  transparent  when  no  water  is 
present.  The  hardening  action  of  the  chloroform  may  be  quickened 
and  itensified  by  carefully  heating  the  chloroform  until  bubbles  of 
ether  begin  to  come  from  the  specimen. 

When  the  collodion  mass  is  hard,  whether  clear  or  not,  pour  off 
the  chloroform  and  add  castor-xylene  clarifier  (§  151),  in  which  the 
tissue  is  to  remain  until  the  sections  are  cut. 

In  a  few  hours  the  collodion  mass  will  become  quite  transparent 
(clarified)  and  hardly  discernable,  so  that  the  tissue  is  readily  seen. 
Sometimes,  however,  the  collodion  remains  white  and  opaque,  due 
to  the  presence  of  moisture,  and  considerable  time  is  required  for  its 


103 

clarification.  In  such  cases  the  process  may  be  hastened  by  placing 
the  tissue  in  the  clarifier  in  a  warm  place,  and  changing  the  clarifier 
several  times.  If  the  block  still  remains  opaque,  remove  to  95% 
alcohol  for  a  day  for  dehydration,  pass  through  chloroform,  and  into 
clarifier.  In  this  way  the  mass  may  usually  be  cleared  perfectly. 
Change  the  clarifier  to  fresh  after  the  first  and  second  days.  The 
sectioning  may  be  done  after  a  few  hours'  immersion,  although  a 
several  days'  clarification  is  preferable.* 

If  a  paper  box  were  used,  after  the  collodion  is  hardened  and 
clarified,  remove  the  paper  box,  absorb  the  castor-xylene  on  the  sur- 
face, trim  the  end  and  put  some  fresh,  thick  collodion  on  the  cork  or 
other  holder.  Press  the  block  firmly  against  the  holder  ;  within  two 
minutes  it  will  be  firmly  cemented  and  one  may  proceed  at  once  to 
clamp  the  holder  in  the  microtome  and  commence  cutting  (§  57). 

§  57.  Cutting  the  sections.  For  collodion  sectioning,  a  long 
drawing  cut  is  necessary  in  order  to  obtain  thin,  perfect  sections. 
The  knife  should,  therefore,  be  set  at  an  obliquity  of  15-20°*  or  less, 
so  that  half  or  more  of  the  blade  is  used  in  cutting  the  section.  Re- 
call that  in  the  paraffin  method  the  knife  is  set  at  right  angles  to  the 
direction  of  the  cut,  and  the  stroke  is  a  rapid  straight  one.  Trim 
away  the  surrounding  collodion  mass,  as  in  sharpening  a  lead  pencil, 
so  that  there  is  not  more  than  a  thickness  of  about  two  millimeters 
all  around  the  tissue,  being  careful,  however,  to  leave  a  broad  base 
as  a  support  to  the  tissue  and  prevent  its  bending  under  the  impact 
of  the  knife  ;  if  the  collodion  mass  is  too  tapering,  bending  will 
occur  and  thin  sections  cannot  be  cut. 

Clamp  the  object  in  the  jaws  of  the  microtome,  placing  it  so  that 
the  mass  of  collodion  is  opposite  the  side  to  which  the  pressure  of  the 
knife  is  applied  in  cutting.  It  is  advantageous  also  to  have  the  ob- 
ject placed  with  its  long  diameter  parallel  with  the  edge  of  the  knife. 

When  knife  and  tissue  are  properly  arranged  wet  the  tissue 
well  with  clarifier  and  flood  the  knife  with  the  same.  Make  the 
sections  with  a  slow,  steady  motion  of  the  knife.  With  a  small  ob- 
ject (3x5  mm.)  and  a  good  sharp  knife,  sections  5jw  to  6yu  can  be  cut 
without  difficulty.  In  addition  to  a  sharp  knife,  however,  there  are 

*The  imbedded  object  may  remain  in  the  castor-xylene  clarifier  indefi- 
nitely without  harm.  The  collodion  grows  somewhat  tougher  by  a  prolonged 
stay  in  it.  After  cutting  all  the  sections  desired  at  one  time,  the  imbedded  tis- 
sue is  returned  to  the  clarifier  for  future  sectioning. 


104 

necessary  well-infiltrated  tissue  and  a  hard,   firm  collodion   mass. 

§  58.  Transferring  the  sections.  If  the  sections  are  quite 
thick  they  may  be  transferred  from  the  knife  to  a  slide  or  a  dish  by 
means  of  forceps  or  a  brush  ;  if  they  are  thin,  however,  it  is  better 
to  handle  them  by  means  of  an  absorbent  tissue  paper,  as  follows  : 
Flood  the  sections  well  with  clarifier  and  then  by  means  of  a  pipette 
remove  the  clarifier  from  the  knife  and  place  over  the  sections  the 
end  of  a  piece  of  the  tissue  paper,  pressing  it  clown  upon  the  sec- 
tions if  necessary.  Carefully  pull  the  paper  off  the  edge  of  the 
knife  ;  the  sections  will  adhere  to  the  paper.  .Place  the  paper,  sec- 
tions down,  on  a  slide,  taking  care  that  the  sections  are  in  the  de- 
sired position.  •  With  the  finger  carefully  press  the  sections  (through 
the  transfer  paper)  to  the  slide,  and  then  lift  the  paper,  with  a  roll- 
ing motion,  from  the  slide  ;  the  sections  will  adhere  to  the  slide. 
Should  they  stick  to  the  paper  instead,  lower  the  paper  again  and 
again  firmly  press  the  section  to  the  slide.  For  further  proceedure 
see  §§  66,  68.  If  it  is  not  desired  to  mount  the  sections  upon  a 
slide  immediately,  or  if  they  are  to  be  kept  in  bulk,  as  for  class 
work,  the  transfer  paper  may  be  shaken  gently  in  a  dish  of  clarifier 
or  95%  alcohol  and  the  section  (or  sections)  will  float  free  and  sink 
to  the  bottom. 

§  59.  If  it  is  desired  to  mount  the  sections  in  a  series,  proceed 
as  follows  :  With  an  artist's  brush  draw  the  first  section,  when  cut, 
up  toward  the  back  of  the  knife  and  make  the  next  section.  Place 
this  section  to  the  right  of  the  first,  and  so  on,  arranging  them  in 
serial  order,  section  after  section,  and  line  below  line,  until  enough 
are  cut  to  fill  the  area  that  the  cover-glass  will  cover.  Flood  the 
sections  as  before  by  letting  the  clarifier  flow  over  them,  being  care- 
ful, however,  not  to  float  them  from  their  places.  Absorb  the  clari- 
fier from  the  knife  with  a  pipette,  and  place  over  the  sections  a 
piece  of  the  transfer  paper  twice  the  width  of  a  slide  ;  press  it  down 
if  necessary,  and  slowly  draw  it  off  the  edge  of  the  knife.  Should 
some  of  the  sections  adhere  to  the  knife  instead  of  the  paper,  it 
means  that  the  clarifier  had  been  allowed  to  thicken*  on  them, 


*  If  one  is  a  long  time  cutting  a  series  of  sections,  it  sometimes  occurs  that 
the  xylene  evaporates  leaving  the  castor  oil  that  is  thick  and  viscid  and  also  a 
solvent  of  the  collodion,  so  that  the  sections  are  not  easily  transferable  but 
stick  rather  firmly  to  the  knife.  In  such  a  case,  fresh  clarifier  or  even  a  little 
xylene  to  dissolve  the  castor  oil  must  be  used. 


105 

cementing  them  to  the  slide,  and  the  preliminary  flooding  to  insure 
their  being  free,  was  insufficient.  In  that  case  it  is  best  to  flood  the 
paper  with  clarifier,  carefully  lift  it,  arrange  the  sections  again,  flood 
them  with  clarifier,  place  a  clean  piece  of  transfer  paper  over  them 
and  try  again.  One  soon  becomes  accustomed  to  the  behavior  of  the 
sections,  and  accidents  are  rare.  In  cutting  a  series  of  many  small 
sections,  some  time  is  consumed  and  it  is  necessary  to  flood  the  sec- 
tions frequently  with  clarifier  while  cutting  in  order  to  prevent  the 
clarifier  thickening  and  cementing  them  to  the  knife. 

§  60.  Resume  of  the  method.  Success  in  the  employment 
of  the  collodion  method  depends  upon  the  thorough  infiltration  with 
the  collodion  solutions,  requiring  days  or  even  months,  and  the  em- 
ployment of  a  thick  imbedding  mass  giving  when  hardened  a  firm 
unyielding  support  to  the  tissue.  This  may  be  gained  by  employ- 
ing a  relatively  long  period  of  infiltration,  and  taking  pains  in  im- 
bedding to  have  the  collodion  mass  well  thickened. 

Observing  these  two  cautions,  collodion  may  be  used  in  almost 
all  cases  as  an  imbedding  mass,  except  such  as  are  affected  by  the 
conditions  of  the  methods  already  mentioned  (§§  40  and  50) . 

THE    FREEZING   METHOD. 

§6 1.  This  method  is  expeditious  and  of  use  in  the  rapid  ex- 
amination of  tissues,  and  therefore  especially  serviceable  in  the 
pathological  laboratory  and  in  clinical  diagnoses.  It  may  also  be' 
used  in  cutting  tissues  that  are  too  hard  to  be  cut  satisfactorily  by 
means  of  either  the  collodion  or  paraffin  methods.  Both  fresh  and 
fixed  tissue  may  be  cut  by  means  of  the  freezing  microtome  and 
with  or  without  any  special  mass  such  as  is  used  in  paraffin  or  collo- 
dion imbedding. 

When  no  mass  is  employed  the  tissue  is  simply  frozen  and  cut, 
or,  if  it  is  fixed  tissue,  soaked  well  in  water  first  and  then  frozen. 
When  extreme  haste  is  not  so  essential  it  is  better  to  first  saturate 
the  tissue  with  some  solution  that  does  not  crystallize  on  freezing, 
but  simply  hardens,  since  the  formation  of  the  ice  crystals  is  hurtful 
to  the  tissue.  Such  are  solutions  of  gum  arabic  or  sugar  and  anise- 
seed  oil,  and  they  are  spoken  or  as  Congelation  masses, 

§  62.  Infiltration.  Gum  arabic  or  anise-seed  oil  may  be 
used,  (a)  Gum  arabic.  If  the  tissue  has  been  fixed  and  is  in  alco- 


io6 

hoi  remove  the  alcohol  by  soaking  it  for  several  hours  to  i  day  in 
water.  Remove  to  a  thick  solution  of  gum  arabic  in  water,  in 
which  it  may  remain  for  about  24  hours.  It  is  then  ready  to  freeze 
and  cut. 

(b)  Anise-seed  oil.  For  this  method  the  tissue  should  be  first 
dehydrated  (§  43).  When  dehydration  is  complete,  transfer  the 
tiSvSue  to  anise-seed  oil,  in  which  it  may  soak  for  12-24  hours  ;  it  is 
then  ready  to  freeze  and  cut. 

§  63.  Cutting.  Place  a  drop  of  the  solution  of  gum  arabic 
(or  anise- seed  oil)  upon  the  object  carrier  of  the  freezing  microtome 
and  turn  on  the  carbonic  acid  (or  ether)  spray.  When  the  mixture 
begins  to  harden,  place  the  object  upon  it  in  an  abundance  of  the 
solution  and  freeze  it  solid.  Covering  with  an  inverted  cup  hastens 
the  freezing. 

When  the  tissue  is  completely  frozen,  cut  it  with  a  straight 
movement  of  the  knife,  as  in  the  paraffin  method,  holding  it  firmly 
upon  the  knife  rest  and  making  the  strokes  as  rapidly  as  possible,  at 
the  same  time  rapidly  raising  the  tissue  a  few  microns  at  a  time  by 
means  of  the  microtome  screw.  For  cutting  frozen  sections,  a 
strong,  wedge-shaped  knife  must  be  used. 

The  mass  of  sections  is  transferred  to  a  dish  of  water  in  which 
the  gum  arabic  is  dissolved  away  and  the  sections  are  ready  for  stain- 
ing (§§  7r~79)-  If  anise-seed  oil  is  used,  the  sections  are  to  be 
transferred  to  95%  alcohol  which  will  dissolve  out  the  oil  ;  stain 
(§§  69-79).  If  the  tissue  has  been  stained  in  toto  the  sections  may 
be  transferred  to  anise-seed  oil  (or  other  clearer)  and  mounted  in 

balsam  directly. 

STAINING  AND  MOUNTING. 

§  64.  The  remaining  steps  in  making  permanent  histological 
preparations  are  usually  done  at  one  time  and  are  conveniently  con- 
sidered together.  In  all  the  processes  seemingly  complicated,  if  it 
is  remembered  that  the  succession  of  media,  as  in  paraffin  imbed- 
ding, depends  on  their  miscibility,  and  the  reason  for  the  various 
steps  is  recognized,  much  of  the  difficulty  in  remembering  the  order 
in  which  they  come  will  be  avoided. 

There  are  here  to  be  considered,  (0)  Paraffin  and  Collodion  sec- 
tions, (b}  Free-hand  and  Frozen  sections,  (r)  Isolation  preparations. 
Of  these,  the  paraffin  and  collodion  sections  require  some  prelimi- 


nary  treatment  not  needed  with  the  others,  and  to  them  only,  there- 
fore, §§  65-70  apply. 


Collodion  Sections 


Paraffin  Sections 


.  V 

fasten  to  slide 


v  v 

not  fastened  to  slide 


fasten  to  slide 


f 

> 
b 

/ 

y 

v 

>rbing  clarifier 

(a)  albumen  fixative 

> 

/ 

:r  alcohol 

(^)  ^Af  %  < 

:ollodion 

v    \ 

1 

f      V 

Benzin 

Alcohol  (95 


Water 


Aqueous  stain 
(hematoxylin) 


Aqueous 
Counter-stain 


Water 


Alcoholic 
Counter-stain 


v 
Alcohol 


Alcoholic  stain 
(Hcl.  carmine) 


v    v    v 

Dehydrate 

(95  %  alcohol) 


V 

Alcohol 


v 
Clear 


Mount  in  balsam 


io8 

PARAFFIN   AND    COLLODION   SECTIONS. 

§65.  These  may  be  carried  on  (a)  as  loose  sections,  or  (b) 
fastened  to  the  slide. 

(a)  Sections  not   fastened  to  the  slide  may  be  carried   on   in 
watch  glasses,  or  larger  glass  vessels  if  there  are  many  of  them,  the 
sections  either  being  transferred  from  vessel  to  vessel  by  means  of 
forceps  or  a  section-lifter,    or  the   fluid  decanted,    care  being  taken 
not  to  pour  off  the  sections,    and  the  succeeding  medium  added. 
Single  sections  may  best  be  carried  on  upon  the  slide,  which   must 
be  kept  horizontal.     When  the  fluid  is  to  be  changed  place  a  brush 
or  needle  gently   on  one  corner  of  the  specimen  and  pour  off  the 
liquid,  if  necessary  first  absorbing  most  of  it  by  means  of  a  pipette  ; 
in  this  way  the  section  may  be  retained  on  the  slide. 

(b)  Fastening  the  Sections  to  the  slide.     This  is  of  great 
advantage  in  carrying  the  sections  on  through   the  different  steps. 
With  serial  sections  it  becomes   an    absolute    necessity.     Different 
methods  are  employed  for  collodion  and  for  paraffin  sections. 

§  66.  (i)  Collodion  sections.  If  the  sections  are  trans- 
ferred to  the  slide  from  clarifier,  absorb  the  oil  thoroughly  by 
placing  over  the  section  some  absorbent  paper  and  pressing  it  down 
gently  and  firmly,  repeating  the  operation  several  times  with  fresh 
paper.  After  the  oil  is  well  absorbed,  with  a  pipette  drop  upon  the 
section  enough  ether-alcohol  to  moisten  it  (1-2  drops).  This  soft- 
ens the  collodion  and  fastens  the  section  to  the  slide.  Allow  the 
ether-alcohol  to  evaporate  until  the  collodion  has  again  set  and  the 
surface  of  the  section  looks  dull  or  glazed,  and  then  place  it  in  ajar 
of  benzin.  Take  care  that  the  specimen  does  not  dry. 

If  the  sections  are  in  series,  it  is  better  to  put  the  ether-alcohol 
on  one  end  of  the  slide  and  let  it  run  quickly  over  the  sections  and 
drain  from  the  other  end  of  the  slide,  repeating  the  operation  two 
or  three  times.  If  it  is  found  that  the  sections  float  off  of  the  slide 
in  the  process  of  staining,  their  adhesion  may  be  insured  by  using 
albumenized  slides  or  removing  the  slides  from  the  alcohol  (§  69) 
and  again  treating  with  ether-alcohol. 

§  67.  (2)  Paraffin  sections.  If  the  sections  are  unwrinkled, 
or  with  a  few  wrinkles  that  may  be  easily  "  ironed  out,"  fasten  them 
to  the  slide  with  (a)  albumin  fixative  and  y±%  collodion,  or  (o) 


io9 

albumin  fixative  and  heat  ;  if  the  sections  are  wrinkled,  they  may 
be  extended  and  the  wrinkles  removed  by  the  method  given  be- 
low (<r). 

(a)  Albumin  fixative   and    ^  %    collodion.      Place   upon    the 
slide  a  small  drop  of  albumin  fixative,  spreading  it  out  with  a  clean 
finger  into  a  very  thin,   even  layer.      Place  the  paraffin  section   in 
position  upon  the  slide  and  with  a  clean  finger,  press  the  section 
into  the  albumin  fixative,  beginning  at  one  edge  of  the  section  and 
by  a  rolling  motion  of  the  finger,  ironing  out  any  wrinkles  there  may 
be.     With  a  camel's  hair  brush  and  by  a  single  sweep,  spread  over 
the  section  a  thin  coating  of  y^%  collodion.     Allow  it  to  dry  in  the 
air  for  a  minute  or  so  and  then  place  the  slide  in  a  jar  of  benzin.     Be 
sure  that  the    y±%  collodion  in  the  bottle  has  not  thickened,  and 
spread  as  little  over  the  section  as  possible,  otherwise  a  much  longer 
time  in  the  benzin  will  be  necessary. 

(b)  Albumin  fixative  and  heat.     The  employment  of  y\%  col- 
lodion causes  the  benzin  to  dissolve  out  the  paraffin  from  the  sections 
slowly,   and  the  collodion  also  stains  intensely  with  certain  dyes, 
such  as  safranin,  gentian  violet,  methylgreen,  methylene  blue,  iron 
hematoxyline,  etc.     When  these  stains  are  to  be  used,  fasten  to  the 
slide  in  the  manner  following  :  Prepare  the  slide  with  albumin  fixa- 
tive and  press  the  section  to  the  slide  as  directed  above.     Look  upon 
the  reverse  side  of  the  slide  to  see  if  the  section  really  adheres  to  the 
albumen  fixative,  as  in  some  cases  it  does  not.     Heat  the  slide  gently 
and  slowly  over  a  small  flame  until  the  paraffin  melts  and  begins  to 
run  away  from  the  specimen.     Keep  the  paraffin  just  melted  for  a 
minute  or  so,  and  then  transfer  to  the  benzin.     A  minute  or  two  in 
benzin   will  suffice.     Should  the  paraffin  section  not  adhere  to  the 
albumin  fixative  when  well  pressed  down,  it  can  in  many  cases  be 
made  to  do  so  by  briskly  rubbing  the  reverse  side  of  the  slide  with  a 
woolen  or  silk  cloth. 

(c)  Extending  sections  with  warm  water.     If  the  sections  are 
wrinkled,  as  they  often  are,  remove  the  wrinkles  by  extending  the 
sections  on  warm  water,  as  followTs  :   Place  the  sections  upon  a  slide 
prepared  with  a  very  thin,  even  layer  or  film  of  albumin  fixative 
(§  67,  «),  and  add  at  the  side  enough  water  to  float  them.     Warm 
the  slide  carefully  and  slowly  over  a  small  flame  until  they  begin  to. 
straighten  out.     Do  not  let  the  water  become  warm  enough  to  melt 


no 

the  paraffin.  When  the  sections  have  become  quite  flat  and  smooth, 
drain  off  the  water  and  allow  the  slide  to  stand  2-3  hours,  or  better 
over  night,  when  a  thin  coating  of  Y\%  collodion  is  given  (§  67,  a} 
and  they  are  placed  in  benzin. 

If  the  sections  are  large  and  thin,  the  coating  with  Y\%  collo- 
dion may  be  omitted  ;  in  this  case,  let  the  sections  stand  12-24  hours 
after  spreading,  or  for  2-3  hours  if  left  in  a  warm  place,  as  in  an 
incubator. 

The  sections  may  also  be  straightened  out  by  placing  them  on 
a  dish  of  water,  such  as  the  waste-jar,  and  hot  water  added  slowly 
until  the  sections  extend,  when,  after  the  water  has  cooled,  they 
may  be  transferred  to  albuminized  slides  and  carried  on  as  before. 

PREPARATION    FOR    STAINING. 

§  68.  Benzin.  Both  paraffin  and  collodion  sections  are  trans- 
ferred to  benzin,  which  in  the  first  case  dissolves  out  the  paraffin,  in 
the  second  serves  to  remove  the  clarifier  still  present  in  the  tissue 
and  collodion. 

Leave  paraffin  sections  in  benzin  until  the  paraffin  is  entirely 
dissolved,  requiring  from  a  minute  or  so  to  10-15  minutes,  if  $% 
collodion  was  used  in  fastening  the  sections  to  the  slide. 

Collodion  sections  should  remain  in  the  benzin  5-15  minutes  or 
longer,  according  to  the  thickness  of  the  sections.  Benzin  may  be 
omitted  and  a  proportionately  longer  time  in  the  alcohol  given. 

Sections  may  be  left  in  benzin  over  night,  and  2-3  days  even 
might  do  no  harm,  but  it  is  better  to  proceed  at  once  to  the  other 
operations. 

§  69.  Alcohol.  Transfer  from  benzin  to  95%alcohol  to  re- 
move the  benzin,  leaving  the  section  in  the  alcohol  5-10  minutes  ; 
or,  if  you  wish,  shorten  the  period  to  a  minute  or  so  by  waving  the 
slide  gently  to  and  fro  in  the  alcohol.  A  stay  of  a  day  or  so  in  alco- 
hol does  no  harm.  Collodion  sections  generally  require  a  longer 
period  than  paraffin  sections  because  of  the  collodion  mass  present 
and  their  greater  thickness. 

§  70.  Water.  Remove  the  95%  alcohol  from  the  section  by 
plunging  the  slide  into  a  jar  of  tap  water  and  gently  waving  it 
about  in  it.  Should  the  stain  to  be  employed  be  an  alcoholic  solu- 
tion this  step  may  be  omitted. 


Ill 

STAINING. 

§  71.  While  staining  has  a  value  in  histology  in  coloring  the 
preparation  as  a  whole  and  thus  making  its  parts  stand  out  more 
sharply,  the  aim  is  to  employ  such  stains  as  by  their  selectivity  give 
different  parts  or  structures  different  colors  or  intensities  of  color, 
thus  differentiating  them  the  better. 

In  histology,  selective  stains  are  employed  (a)  to  color  the 
nucleus  differentially,  thus  distinguishing  it  sharply  from  the  cell 
body,  and  by  means  of  the  nucleus  setting  apart  groups  of  cells  ; 
(b)  to  distinguish  between  different  kinds  of  tissue,  as,  for  example, 
connective  tissue  and  muscle. 

§  72.     The  following  two  general  rules  may  be  given  : 

1 i )  The  preparation  should  be  transferred  to  the  stain   from 
the  fluid  in  which  the  staining  principle  is  dissolved.     The  majority 
of  the  stains  employed  are  aqueous  solutions  ;  some,  however,  such 
as   hydrochloric  acid  carmine,  are  alcoholic  ;  with  these  alcoholic 
stains  the  last  preparatory  step   (§  70),  removal  of  the  alcohol  by 
water,  should  be  omitted.     Many   stains   may    be   used   either   in 
aqueous  or  in  alcoholic  solution. 

(2)  The  stain  should  be  washed  out  with  the  solvent  of  the 
dye, — water  or  alcohol  as  the  case  may  be. 

With  many  aniline  dyes  (§  74)  one  may,  and  in  many  cases 
should,  employ  alcohol  for  washing  out,  even  though  the  staining 
solution  is  aqueous. 

§  73.  Differentiation.  To  bring  out  the  differential  or 
selective  coloring  of  the  dye.  This  is  necessary  with  many  stains, 
especially  anilines,  and  may  often  be  satisfactorily  accomplished  by 
the  use  of  95%  alcohol  and  made  a  part  of  the  washing  out.  Alcohol 
acidified  with  hydrochloric  acid  (§  149)  is  sometimes  used,  or  a 
special  differentiator  is  required. 

§  74.  Classification.  (#)  According  to  their  chemical  nature 
the  stains  generally  used  are  (i)  hematoxylin  stains,  (2)  carmine 
stains,  (3)  aniline  stains 

(d)  According  to  their  selectivity.  Stains  as  used  in  histology 
may  for  convenience  be  classified  as  (i)  nuclear,  (2)  general,  and 
(3)  special.  These  are  often  used  in  combinations  of  two  or  three, 
either  together  in  the  same  solution  or  separately  in  solutions  used 


112 

successively.  Double  and  triple  stains  are  usually  combinations  of 
nuclear  with  general  or  special  stains.  Quadruple  stains  are  less 
serviceable  ;  see,  however,  p.  68,  154. 

(c)  According  to  the  mode  of  employment,  staining  may  be 
either  (i)  in  toto,  or  (2)  section  staining.  When  in  toto  stain- 
ing is  employed  the  piece  of  tissue  is  stained  entire,  and  imbedded 
and  sectioned  afterwards.  In  this  case  the  tissue  should  be  stained 
before  the  process  of  imbedding  is  begun,  after  the  washing  out  of 
the  fixer  has  been  completed  (§  19,  21).  But  a  single  stain  may  be 
given,  and  the  one  chosen  is  generally  a  nuclear  one — hematoxylin 
or  carmine. 

(2)  Section  staining,  i.  e.,  after  the  tissue  is  imbedded  and  sec- 
tions cut.  This  is  more  serviceable,  especially  if  highly  differential 
results  are  desired. 

§  75.  Impregnations.  In  addition  to  the  typical  methods  of 
coloring  tissue  by  means  of  stains  there  is  a  group  of  methods  in 
which  the  coloring  matter  is  deposited  in  the  cell  or  tissue  that  it  is 
desired  to  differentiate,  in  the  form  of  a  precipitate.  These  are 
known  as  impregnation  methods  and  are  of  great  value,  especially 
as  applied  to  nervous  tissues.  A  hard  and  fast  line,  however,  can- 
not be  drawn  between  true  staining  methods  and  impregnations. 
Silver  nitrate  and  gold  chlorid  are  the  substances  most  generally 
employed  in  the  impregnation  of  tissue  (§  146). 

§  76.  Choice  of  stains.  Remember  that  the  staining  is 
greatly  affected  by  the  previous  treatment  ;  brilliancy  or  selectivity 
in  the  result  being  in  many  cases  dependent  on  the  fixer  employed 
or  impaired  by  the  improper  or  incomplete  washing  out  of  the  fixer. 

In  staining,  therefore,  consider  three  things,  (a)  what  it  is  de- 
sired to  bring  out, — what  kinds  of  stains  you  need  to  employ,  (ft) 
the  mode  of  fixation  that  has  been  employed,  and  (V)  the  imbedding 
method  must  also  be  considered  in  the  choice,  since  if  collodion  is 
employed  certain  stains  that  color  it  deeply  should  be  avoided. 

§  77.  The  time  of  staining.  Although  in  general  certain 
time  limits  can  be  given  to  the  period  during  which  a  stain  should 
be  allowed  to  act  ;  with  most  stains,  especially  those  with  which  no 
differentiation  is  needed,  such  as  hematoxylin,  and  most  carmines, 
the  correct  intensity  of  color  should  be  determined  by  examining  the 
preparation  with  the  microscope.  One  soon  becomes  able  to  judge 


of  the  right  stain  in  this  way  better  than  if  a  given  time  were  ad- 
hered to. 

In  the  use  of  stains  requiring  a  subsequent  differentiation,  the 
rule  is  to  over^stain  and  watch  the  differentiation  carefully  with  the 
microscope,  stopping  it  when  sufficient.  In  this  case  it  is  the  differ- 
entiation and  not  the  staining  that  should  be  carefully  regulated. 

§  78.  In  this  course  are  employed  :  (i)  Chloral  hematoxylin, 
(2)  Ehrlich's  acid  hematoxylin,  (3)  Delafield's  hematoxylin,  (4) 
Weigert's  hematoxylin,  (5)  Borax  carmine,  (6)  Alum  carmine 
(Grenacher's),  (7)  Paracarmine,  (8)  Hydrochloric  acid  carmine, 
(9)  Picro-carmine,  (10)  Safranin,  (n)  Gentian  violet,  (12)  Methyl- 
green,  (13)  Ehrlich's  triacid  mixture,  (14)  Eosin,  (15)  Erythrosin, 
(16)  Picric  alcohol,  (17)  Picro-fuchsin  ;  also  (18)  Heidenhain's 
iron  hematoxylin,  and  special  stains  given  subsequently  under 
"Special  Methods." 

§  79.  The  combinations  most  employed  are  (a)  hematoxylin 
and  eosin  (or  erythrosin),  and  (^)  hematoxylin  and  picrofuchsin, 
the  former  a  double  and  the  latter  a  triple  combination. 

The  following  scheme  indicates  the  manner  of  their  employment : 


Paraffin  or  Collodion  Sections 
(§§66  and  67) 


v 
Benzin 


95  %  Alcohol 


Water 


v 
Hematoxylin,  10-30  min. 


v 
Water 


v 

Picrofuchsin.  eosin 
or  erythrosin,  ^2-2  min. 


v 
Distilled  water  or  67  %  alcohol 


v 
Dehydrate  (95-99  %  alcohol) 


Clear 


(carbol-xylene) 


y 
Mount  in  Balsam 


STAINS. 

|  80.  Chloral  Hematoxylin.  (Proc.  Am.  Micr.  Soc.,  vol.  XIV,  pp.  125- 
127).  Formula:  (A)  Hematoxylin  crystals,  -fa  gram,  dissolved  in  10  c.c.  of 
95%  alcohol  ;  (B)  distilled  water,  100  c.c.,  potash  alum,  3^  grams  ;  boil  for  5 
minutes  in  an  agate-ware  or  glass  vessel  and  add  sufficient  boiled  water  to  bring 
the  volume  back  to  100  c.c.  When  cool,  mix  (A)  and  (B)  and  add  chloral  hy- 
drate, 2  grams.  Allow  the  solution  to  ripen  for  a  week  or  two  in  the  air  and 
sunlight,  or  add  1-2  c.c.  hydrogen  dioxid,  when  it  is  ready  for  use.  Its  staining 
quality  improves  up  to  an  optimum  and  then  begins  to  deteriorate.  Old 
hematoxylin  generally  contains  a  precipitate  and  should  be  filtered  often  or 
before  using. 

Stain  sections  from  water  for  5.30  minutes  according  to  the  age  of  the  solu- 
tion, the  character  of  the  tissue  and  the  fixation  employed.  After  staining, 


wash  well  with  tap  water  to  give  a  blue  tone  to  the  stain.  Counter-stain  as 
desired. 

$  81.  Ehrlich's  acid  hematoxylin.  Formula:  Water,  looc.c.  ;  95%  alco- 
hol, loo  c.c.  ;  glycerin,  100  c.c.  ;  glacial  acetic  acid,  10  c.c.  ;  alum  in  excess. 
L/et  the  mixture  ripen  in  the  light  until  a  dark  red.  Sections  stain  in  this  hem- 
atoxylin in  a  short  time,  generally  5-10  minutes.  Wash  with  water  after 
staining. 

$  82.  Delafield's  hematoxylin.  Formula :  Saturated  aqueous  solution  of 
ammonia  alum,  200  c.c.  ;  16%  alcoholic  solution  of  hematoxylin  crystals,  12^ 
c.c.  (hematoxylin  crystals  2  grams,  95%  alcohol  12%  c.c.).  Allow  the  mix- 
ture to  stand  in  the  light  and  air  in  an  unstoppered  bottle  for  4  or  5  days  ;  filter 
and  add  glycerin,  50  c  c.,  and  methylic  alcohol,  50  c.c.  Permit  it  to  stand  for 
a  week  or  so  to  ripen  ;  filter  and  keep  in  a  stoppered  bottle.  The  staining  power 
increases  for  several  months.  In  using,  dilute  3  or  4  times  with  distilled  water. 

Stain  sections  from  water  ;  4-5  minutes  will  generally  be  sufficient.  Wash 
well  with  water  after  staining. 

This  is  a  very  strong  hematoxylin  stain  and  may  be  used  to  advantage  with 
tissues  that  stain  with  difficulty.  It  is  likewise  a  more  diffuse  stain  than  either 
chloral  or  Ehrlich's  hematoxylin,  staining  cell-body  as  well  as  nucleus, — a  fea- 
ture having  its  advantages  and  disadvantages.  Old  solutions  (several  months 
to  a  year )  should  be  filtered  before  using. 

$  83.  Weigert's  hematoxylin.  Formula  :  Hematoxylin,  i  gram,  dis- 
solved in  95%  alcohol,  10  c.c.  ;  distilled  water,  90  c.c.  ;  saturated  aqueous  solu- 
tion of  lithium  carbonate,  i  c.c.  This  stain  may  be  used  at  once  or  (better)  be 
allowed  to  ripen  for  2-3  days. 

Tissue  should  be  stained  from  water  and  rinsed  in  water  after  staining. 
This  is  a  powerful  stain  and  is  used  in  Weigert's  method  of  staining  myelinic 
nerve  fibers  ( '4.  1 36 ) . 

\  84.  Borax  carmine.  (Grenadier).  Formula:  Borax,  4  grams;  car- 
mine, 3  grams  ;  water,  100  c.c.  ;  allow  the  mixture  to  stand  for  several  days, 
shaking  occasionally  when  most  of  the  carmine  will  have  dissolved  ;  filter  and 
add  100  c.c.  of  70%  alcohol.  Let  the  mixture  remain  for  several  days,  filter 
again  again  and  the  solution  is  ready  for  use. 

This  is  a  good  carmine  stain  for  in  toto  staining.  Stain  objects  in  toto  for 
one  to  several  days,  according  to  size;  remove  to  67%  (jo%)  alcohol,  acidu- 
lated slightly  with  hydrochloric  acid,  (4  drops  in  each  100  c.c.),  for  a  day  and 
then  remove  to  82%  alcohol.  It  affords  a  bright  red  stain  that  is  quite  trans- 
parent. 

g  85.  Alum  carmine.  (Grenacher).  Formula:  Carmine,  I  grm  ;  potash 
(or  ammonia)  alum,  4  grams  (1-5  grams)  ;  water,  100  c.c.  ;  boil  for  15-20  min- 
utes, allow  it  to  cool  and  filter. 

A  good  carmine  stain  for  sections.  Stain  5-30  minutes,  or  longer  if  neces- 
sary ;  rinse  off  the  excess  of  stain  with  distilled  water.  The  stain  is  a  purple- 
red. 


u6 

\  86.  Paracarmine  (Mayer).  Formula:  Carminic  acid,  i  gram  ;  alumi- 
num chlorid,  .5  gram  ;  calcium  chlorid,  4  grams  ;  70%  (67%)  alcohol,  100  c.  c. 
Allow  it  to  stand  a  day  or  so,  shaking  occasionally  until  the  carminic  acid  has 
quite  dissolved,  and  then  filter. 

This  is  an  excellent  carmine  stain  for  in  toto  staining.  The  tissue  may  be 
stained  i-several  days  (i  week),  then  washed  in  67%  and  82%  alcohols  to  re- 
move the  excess  of  staining  fluid.  A  red  nuclear  stain,  more  opaque  than 
borax  carmine.  It  does  not  over-stain  readily,  and  since  it  is  an  alcoholic  so- 
lution (70%)  it  is  quite  penetrating  and  may  be  allowed  to  act  for  a  greater 
length  of  time,  being  thus  suited  for  staining  in  toto  objects  of  considerable 
size. 

\  87.  Hcl.  carmine.  Formula  :  Carmine,  2  grams  ;  concentrated  hydro- 
chloric acid,  3  c.  c. ;  70%  alcohol,  100  c.  c.  Boil  gently  for  15-20  minutes  to 
dissolve  the  carmine  ;  cool  and  filter. 

This  is  a  strong  carmine  stain,  quite  suitable  for  sections,  especially  such 
as  stain  with  difficulty.  It  may  also  be  employed  for  staining  in  toto.  Stain 
sections  from  alcohol  or  water  for  5-15  minutes  ;  rinse  away  the  superfluous 
stain  with  67%  (70%)  alcohol  and  differentiate  for  a  few  seconds  to  a  minute 
with  acid  alcohol  (95%  alcohol  looc.  c. ,  concentrated  hydrochloric  acid  i  c.  c.). 
Wash  away  the  acid  alcohol  with  ordinary  95%  alcohol.  If  a  pure  nuclear 
stain  is  not  desired  the  differentiation  may  be  omitted. 

Picric  alcohol  may  be  used  as  a  counter  stain,  and  in  that  case  differentia- 
tion is  ordinarily  not  required. 

\  88.  Picro-carmine.  Employ  a  %%  aqueous  solution  of  the  dry  picro- 
carmine,  made  according  to  Ranvier's  formula.  Stain  sections  J^-i  hour  or 
longer  and  rinse  well  in  water.  If  the  sections  are  over-stained  they  may  be 
differentiated  by  means  of  acid  alcohol  as  used  with  Hcl.  carmine. 

While  it  is  itself  a  good  stain,  it  may  be  used  with  hematoxylin  as  a  coun- 
ter-stain with  good  results.  It  is  an  alkaline  solution,  and  the  hematoxylin 
stain  is  intensified.  As  employed  in  this  way,  stain  in  hematoxylin  the  usual 
length  of  time,  rinse  in  water  and  stain  2-3  hours  in  the  picro  carmine  solution  ; 
rinse  in  water ;  dehydrate,  clear,  and  mount  in  balsam.  As  used  in  this  way, 
there  is  afforded  an  excellent  stain  for  developing  bone. 

\  89.  Safranin.  Formula  (Babes')  :  Concentrated  aqueous  solution  of 
safranin,  i  part ;  concentrated  alcoholic  solution  of  safranin,  i  part. 

Stain  sections  1-4  hours,  or  over  night ;  wash  away  excess  of  stain  with  95% 
alcohol,  differentiate  with  acid  alcohol  (95%  alcohol,  100  c.c.,  hydrochloric 
acid,  rV  c.c. )  for  a  few  seconds,  rinse  with  95%  alcohol  and  clear  in  carbol- 
xylene  or  bergamot  oil.  If  a  pure  nuclear  stain  is  not  required,  the  differentia- 
tion may  be  omitted.  This  gives  a  good  stain  with  tissue  fixed  in  Hermann's 
or  Flemming's  fluid.  It  is  a  brilliant,  transparent  red.  (See  \  99). 

\  90.  Gentian  violet.  Formula :  A  concentrated  solution  in  distilled 
water.  Stain  (paraffin)  sections  from  water  for  5-10  minutes,  rinse  in  water, 
dehydrate  and  differentiate  with  95%  alcohol  and  complete  the  differentiation 
with  clove  oil.  When  the  differentiation  is  sufficient,  clear  with  bergamot  oil 


and  mount  in  balsam.  This  may  be  used  alone  to  give  a  blue  stain  with  tissue 
fixed  in  Hermann's  or  Flemming's  fluid. 

$  91.  Methyl  green.  This  is  an  nuclear  stain  of  much  value,  besides 
being  an  important  ingredient  of  triple  stains  (e.g.,  Ehrlich's  triacid  mixture). 
In  very  dilute  solutions  it  is  serviceable  in  staining  the  nuclei  of  fresh  tissue 
and  of  isolated  cells  (formula  below).  A  i%  aqueous  solution  is  used  with 
hematoxylin  and  picrofuchsin  in  differentiating  the  structure  of  the  hair  folli- 
cle. (See  1 54). 

£  92.  Methyl  green  and  eosin.  Formulas:  (a)  Aqueous  solution.  i% 
aqueous  solution  of  methyl  green,  \y2  c.c.  ;  %%  aqueous  solution  of  eosin,  i 
c.c.  ;  normal  salt  solution,  100  c.c. 

(b)  Glycerin  solution  (for  mounting).  i%  aqueous  solution  of  methyl 
green,  2  c.c.  ;  V2%  aqueous  solution  of  eosin,  I  c.c.  ;  glycerin,  100  c.c. 

These  solutions  may  be  used  for  staining  isolated  cells  ;  formula  (a)  for 
temporary  examination,  formula  (b)  as  a  mounting  and  staining  medium 
combined. 

$  93.  Ehrlich's  triacid  mixture.  Formula :  Saturated  aqueous  solution 
of  orange  G,  12  c.  c. ;  saturated  aqueous  solution  of  acid  fuchsin,  8  c.  c.;  sat- 
urated aqueous  solution  of  methyl  green,  10-12  c.  c.;  distilled  water,  30  c.  c. ; 
alcohol  (95%  -j- ),  18  c.  c. ;  glycerin,  5  c.  c.  Mix  the  orange  G  and  acid  fuchsin 
and  add  drop  by  drop,  or  a  few  drops  at  a  time,  the  solution  of  methyl  green, 
stirring  or  shaking  between  each  addition  ;  then  add  the  alcohol,  water,  and 
glycerin.  Shake  thoroughly  and  allow  the  mixture  to  stand  for  24  hours.  Do 
not  filter  or  shake,  but  take  the  stain  from  the  bottle  by  means  of  a  pipette. 

Stain  sections  for  10-15  minutes,  rinse  off  the  superfluous  stain  with  dis- 
tilled water  and  dehydrate  and  clear  as  quickly  as  possible  ;  this  is  necessary 
since  the  alcohol  washes  out  the  methyl  green  (nuclear  stain)  very  rapidly. 
This  stain  colors  collodion  very  deeply,  hence  it  cannot  well  be  used  with  col- 
lodion sections.  It  affords  a  good  and  often  valuable  stain.  The  sections 
should,  however,  beihin,  and  dehydration  must  be  rapid.  For  the  use  of  the 
mixture  in  staining  blood  films,  see  \  125. 

$94.  Eosin.  Formulas:  (a)  y2%  aqueous  solution;  (b)  2%  aqueous 
solution  ;  (c)  ^%  solution  in  water  or  50%  alcohol.  Formula  (a)  is  prefera- 
ble for  most  work;  (b)  affords  a  stronger  and  (c)  a  weaker  stain.  This  may 
be  used  as  a  counter-stain  with  hematoxylin  to  differentiate  nucleus  from 
cell-body.  Stain  sections  after  hematoxylin  for  10-30  seconds,  wash  away  the 
excess  of  stain  with  distilled  water  or  6j%  alcohol.  Since  alcohol  tends  to 
wash  out  the  eosin,  unless  the  color  is  too  strong  it  is  advisable  to  hasten  the 
process  of  washing  out  and  dehydration. 

^95.  Erythrosin.  Formulas:  (a)  l/2-i%  solution  in  67%  alcohol,  (b) 
y2-\%  aqueous  solution,  ( c)  ^%  aqueous  solution.  This  is  a  general  stain  sim- 
ilar to  eosin  in  its  staining  properties,  but  gives  a  redder  color.  Formulas  (a), 
and  (b)  may  be  used  with  sections  and  in  the  same  way  as  eosin.  Formula  (c) 
is  used  with  tissues  dissociated  in  formaldehyde  dissociator. 

\  96.     Picric   alcohol    (acid).     Formula:    Picric  acid,  .2  grams;  distilled 


u8 

water,  500.0.  ;  95%  alcohol,  50  c.c.  A  good  counter-stain  with  hematoxylin 
or  carmine.  Stain  in  hematoxylin  or  carmine,  rinse  in  water  (or  alcohol  if  Hcl. 
carmine  is  used),  and  stain  with  picric  alcohol  10-30  seconds  ;  wash  off  with 
67%  alcohol,  etc.  Since  picric  acid  tends  to  wash  out  the  nuclear  stain,  it  is 
best  to  over-stain  somewhat  with  the  nuclear  dye  and  regulate  carefully  the 
time  of  staining  with  the  picric  alcohol. 

|  97.  Picro-fuchsin.  Formulas:  (a)  General  stain, — 1%  aqueous  solu- 
tion of  fuchsin  acid,  10  c.  c.;  saturated  aqueous  solution  of  picric  acid,  75  c.c.  ; 
distilled  water,  25  c.c.  (b)  For  nervous  tissue, — i%aqueous  solution  of  fuchsin 
acid,  15  c.c.  ;  saturated  aqueous  solution  of  picric  acid,  50  c.c.  ;  distilled  water, 
50  c.c.  This  is  a  valuable  counter-stain  to  hematoxylin,  especially  serviceable 
in  the  differentiation  of  white  connective  tissue  fibers.  The  nuclei  are  a  pur- 
plish brown  (hematoxylin  stain),  the  connective  tissue  red,  cell  bodies  and 
muscle  yellow-orange.  In  special  cases  the  relative  amount  of  fuchsin  acid 
may  be  decreased  or  increased,  thus  giving  a  preponderance  to  the  yellow  or 
red  in  the  general  stain. 

Stain  well  with  hematoxylin,  rinse  in  water,  and  stain  with  the  picro-fuch- 
sin  15-30  seconds  ;  wash  away  the  excess  of  stain  with  distilled  water  or  67% 
alcohol.  Picro-fuchsin  will  gradually  wash  out  the  hematoxylin,  therefore 
stain  strongly  with  hematoxylin  and  regulate  carefully  the  time  of  staining 
with  picro-fuchsin.  Picro-fuchsin  is  quite  sensitive  to  alkalies,  so  that  tap-water 
(unless  slightly  acidulated )  should  not  be  used  for  washing  out,  and  the  mount- 
ing medium  should  be  slightly  acid  or  neutral,  not  alkaline. 

$  98.  Heidenhain's  Iron  Hematoxylin.  Three  steps  are  necessary,  (a) 
mordanting,  (b)  staining,  (c)  differentiating. 

1.  Mordant  sections  1-2  hours  in  a  4%  aqueous  solution  of  ferric  alum 
(iron-ammonium-persulphate),  rinse  in  water  a  few  (5-10)  minutes,  and 

2.  Stain  for  1-3  hours  in  a  Yz%   aqueous  solution   of  hematoxylin  ( form- 
ula :     1 6%  alcoholic  solution  of  hematoxylin  30.  c.,  water  97  c.  c. ,  chloral  hy- 
drate 2  grams),  rinse  in  water,  and 

3.  Differentiate  by  dipping  the  slide  into  the  mordant  for  a  few  seconds 
and  then  rinsing  in  tap-water,  repeating  the  operation  until  the  correct  differ- 
entiation has  been  attained,  as  ascertained  by  examination  under  the  micro- 
scope.    The   chromatin   (nucleus)  should  be  a  deep  blue  or  blue-black,  the 
cytoplasm  ( cell-body )  gray  or  light  blue.     After  differentiating  it  is  necessary 
to  wash  the  section  well  in  running  water  for  about  20  minutes  to  ensure  the 
complete  removal  of  the  ferric  alum.     The  above  times  are  for  tissue  fixed  in 
Flemming's  or  Hermann's  fluid  ;  shorter  times  may  be  given  for  tissue  fixed 
by  most  other  methods. 

A  counter-stain  is  ordinarily  not  needed,  but,  if  desired,  the  sections  may 
be  stained  for  10  seconds  to  2-3  minutes  with  a  y2  saturated  aqueous  solution  of 
orange  G. 

This  is  a  valuable  stain  in  all  cytological  work,  and  for  all  muscular  tissue. 


Iron  Hematoxylin 

Any  Fixation  desired 

v 
Paraffin  Sections 


v 
Benzin 


95.%  Alcohol 


Water 

I 

v 

Mordant 
1-3  hours  in  4  %  ferric  alum 


WaVter 


v 

Stain  1-6  hours 
in  V<2  %  aq.  hematoxylin 


Water 


V 

Differentiate,  by  dipping  alternately  in 

mordant  and  in  the  tap-water, 

a  second  in  each 

I 

v 

Water  (running) 
15  minutes 
I  or 


v 

Orange  G. 
sat.  aq.  sol.) 


v 
Water  (rinsed) 


>  95-99  %  Alcohol 


v 
Clear  in  Carbol-xylene 

I 

v 
Mount  in  Balsam 


120 


§  99-  Staining  with  Safranin 

Flemming's  or  Hermann's 
fluid  fixation  (best) 


v 
Paraffin  sections 

i 

v 
Benzin 

i 

v 
95  %  Alcohol 

I 

v 

Safranin  (Babes') 
2-12  hours 

i 

v 

Distilled  water 
(rinsed) 

[ 

95  %  Alcohol 
| 

Acid  v$%  alcohol  (TV  Hcl.) 
for  a  few  seconds 

i 

v 
95-99%  Alcohol 

i 

v 
Carbol-xylene 

v 
Mount  in  Balsam 

MOUNTING. 

§  loo.  Whether  stained  or  unstained,  prepared  for  microscop- 
ical examination  by  isolation  or  sectioning,  and  especially  if  it  is  de- 
sired to  keep  the  preparation,  it  is  necessary  to  mount  it  in  some 
way, — i.  e.,  so  arrange  it  upon  some  suitable  support  (glass  slide) 
and  in  some  suitable  mounting  medium  that  it  may  be  satisfactorily 
studied  with  the  microscope. 
Mounting  may  be 

I.     Temporary,  or 
II.     Permanent, — as 

A.  Dry,  or  in  air, 

B.  In  a  medium  miscible  with  water,  or 

C.  In  a  resinous  medium,  in  which  case  it  is  neces- 


121 

sary  first  to  remove  all  water  by  either  (a)  drying — Desiccation,  or 
(d)  a  series  of  displacements,  i.e.,  i.  Removing  the  water  with 
strong  alcohol — Dehydration;  2.  Removing  the  alcohol  with 
clearer — Clearing  ;  3.  Replacing  the  clearer  with  balsam  or  other 
resinous  mounting  medium. 

§  10 1.  Temporary  mounting.  Used  in  this  course  princi- 
pally in  the  examination  of  blood  corpuscles  and  living  ciliated  cells 
(§2).  Temporary  examination  of  tissues  is  quite  simple,  though 
important,  and  for  this  it  is  only  necessary  to  place  the  teased  tissue 
or  section  on  the  slide  in  a  drop  of  the  fluid  in  which  it  is  at  the 
time,  normal  salt  solution,  dissociator,  or  alcohol,  and  cover.  The 
examination  of  preparations  intended  for  permanent  mounts  during 
the  staining  or  before  mounting  will  often  serve  to  detect  faulty 
treatment  at  a  time  when  it  may  be  remedied  without  great  expend- 
iture of  time,  or  discard  the  specimen  as  worthless. 

§  102.  Permanent  mounting.  In  this  course  are  employed 
(«)  mounting  dry  on  a  ring  or  in  a  cell,  (£)  in  glycerin  or  glycerin 
jelly,  media  miscible  with  water,  and  (c^)  in  xylene  balsam,  a  resin- 
ous medium. 

§  103.  Mounting  dry.  The  preparation  may  be  either  upon 
the  under  side  of  the  cover-glass  (best  if  possible)  or  rest  upon  the 
bottom  of  the  cell. 

In  the  first  case  a  shallow  cell  made  by  a  shellac  ring  will  be 
sufficient  ;  in  the  second,  a  shellac  ring  may  not  give  a  deep  enough 
cell  and  a  paper,  hard  rubber,  or  metal  ring  may  be  cemented  to  the 

slide. 

(a)  When  the  preparation  is  on  the  cover.     Prepare  a  shellac 

cell  (§  1 06)  on  the  slide  of  a  size  slightly  smaller  than  the  cover  to 
be  used,  and  allow  it  to  dry  for  a  day  or  so.  Warm  the  cover  bear- 
ing the  preparation  to  remove  the  last  traces  of  moistuie,  and  place 
it  film  side  down  upon  the  ring.  Warm  the  slide  until  the  edge  of 
the  cover  may  be  made  to  adhere  to  the  shellac  ring  and  press  the 
cover  down  until  it  adheres  all  the  way  round.  Seal  the  cover  with 
shellac  and  label  (§§  in,  113). 

(b)  Mounting  in  a  paper  or  rubber  cell.     With  a  brush,  cover 
one  side  of  the  ring  with  a  layer  of  shellac  and  place  it  on  the  center 
of  the  slide,  shellac  side  down  ;  place  within  the  cell  the  prepara- 
tion, arranging  it  in  the  manner  desired,  and  place  upon  the  ring  a 
cover-glass  of  a  suitable  size,  and  seal  it  with  shellac  ;  label. 


§  104.  Mounting  in  glycerin  media,  (a)  Pure  glycerin  ; 
(b)  glycerin  and  acetic  acid,  i  %;  (V)  glycerin,  alum  carmine  and 
eosin  (§  156). 

Glycerin  and  glycerin-jelly  are  most  serviceable  in  mounting 
isolation  preparations.  For  both  of  these  mounting  media  the  ob- 
ject must  be  mounted  from  water  or  an  aqueous  solution. 

Arrange  the  section  or  teased  tissue  in  the  center  of  the  slide, 
drain  off  the  water  or  aqueous  solution  in  which  the  preparation  is 
and  add  a  small  drop  of  glycerin.  Take  a  clean  cover  in  the  forceps, 
breathe  on  the  under  side  and  carefully  lower  it  upon  the  object  ; 
gently  press  it  down.  It  is  best  to  use  only  a  small  drop  of  glycerin 
so  as  not  to  get  it  outside  the  cover,  as  it  is  hard  to  clean  away  sat- 
isfactorily. Clean  carefully  and  seal  with  shellac  in  accordance 
with  §111. 

§  105.  Mounting  in  glycerin-jelly.  The  preparation  should 
be  mounted  from  some  aqueous  solution.  Warm  the  slide  gently 
and  put  it  upon  the  centering  card  ;  in  the  center  of  the  slide  place 
a  drop  of  warmed  (melted)  glycerin-jelly.  Remove  the  object  from 
the  water  or  aqueous  solution  and  arrange  it  in  the  glycerin -jell)7. 
Grasp  a  cover-glass  with  the  fine  forceps,  breathe  on  the  lower  side, 
gradually  lower  it  upon  the  object  and  gently  press  it  down.  Allow 
the  glycerin-jelly  to  set,  keeping  the  slide  horizontal  meanwhile. 
Scrape  away  the  superfluous  glycerin:jelly  around  the  cover-glass 
and  seal  with  shellac  (§  in). 

§  106.  Preparation  of  shellac  mounting  cells.  Place  the 
slide  upon  the  turn  table  and  center  it  (i.  e.,  get  the  center  of  the 
slide  over  the  center  of  the  turn  table).  Select  a  guide  ring  on  the 
turn  table  which  is  a  little  smaller  than  the  cover-glass  to  be  used  ; 
take  the  brush  from  the  shellac,  being  sure  that  there  is  not  enough 
cement  adhering  to  it  to  drop.  Whirl  the  turn  table  and  hold  the 
brush  lightly  on  the  slide  just  over  the  guide  ring  selected.  An 
even  ring  of  the  cement  should  result.  If  it  is  uneven,  the  cement 
is  too  thick  or  too  thin  or  too  much,  was  on  the  brush.  After  a  ring 
is  thus  prepared,  remove  the  slide  and  allow7  the  cement  to  dry 
spontaneously,  or  heat  the  slide  in  some  way.  Before  the  .slide  is 
used  for  mounting,  the  cement  should  be  so  dry  when  it  is  cold 
that  it  does  not  dent  when  the  finger  nail  is  applied.  A  cell  of  con- 
siderable depth  ma)7  be  made  with  shellac  by  adding  successive  lay- 
ers as  the  previous  one  dries. 


123 

§  107.  Mounting  in  balsam.  Certain  preparations  may  be 
mounted  in  balsam  by  drying,  the  method  of  desiccation  (§  100),  e. 
g.,  cover- glass  preparations  of  bacteria,  stained  cover-glass  prepara- 
tions of  blood,  etc.  For  this  it  is  only  necessary  that  the  prepara- 
tion be  absolutely  dry,  a  small  drop  of  balsam  placed  upon  it  or 
upon  the  under  side  of  the  cover- glass,  which  is  carefully  placed 
over  the  specimen  and  pressed  down. 

Mounting  in  balsam  by  desiccation  is  serviceable  for  but  few 
preparations  in  histology,  and  in  most  cases  the  removal  of  the 
water  by  a  series  of  displacements  is  resorted  to  (§  100).  For  this 
the  following  steps  are  necessary  :  Dehydration,  Clearing,  Mount- 
ing in  balsam. 

Dehydration.  The  sections  are  entirely  freed  from  water  by 
the  use  of  95%  or  absolute  alcohol.  The  slide  or  free  section  may 
either  be  placed  in  a  jar  of  alcohol  or  alcohol  from  a  pipette  be 
poured  over  it.  Treat  the  preparation  to  be  mounted  for  5-15  min- 
utes. The  thicker  the  section  the  longer  the  time  required  ;  collo- 
dion sections  require  a  longer  time  than  paraffin  sections.  In  any 
case,  be  sure  that  the  dehydration  is  complete,  giving  a  longer 
rather  than  a  shorter  time,  and  then  clear. 

§  108.  Clearing.  This  is  accomplished  by  putting  the  slide 
in  a  jar  of  clearer  or  dropping  the  clearer  upon  the  section  from  a 
pipette.  When  the  section  is  cleared  it  will  be  transparent.  Test 
it  by  holding  it  against  a  dark  background  ;  if  it  is  not  cleared  it 
will  be*  cloudy,  white,  and  opaque. 

§  109.  Mounting  in  balsam.  Drain  off  the  clearer  and  allow 
the  section  to  stand  until  there  appears  the  first  sign  of  dullness 
from  evaporation  of  the  clearer  from  the  surface.  Then  place  a  small 
drop  of  balsam  upon  the  section  or  upon  the  cover-glass  which  is 
then  inverted  over  the  specimen. 

Remember  that  in  mounting  in  this  way  you  must  always  ' '  De- 
hydrate, Clear  and  Mount  in  Balsam"  and  that  the  three  steps 
are  inseparable. 

SEALING   THE   PREPARATIONS. 

This  is  only  necessary  when  the  preparation  is  a  glycerin  or 
glycerin-jelly  mount.  It  is  better  not  to  seal  balsam  preparations, 
or  only  quite  late  after  the  balsam  has  thoroughly  dried  out. 


124 

§  no.  Sealing  glycerin  mounted  specimens.  Wipe  away 
the  superfluous  glycerin  as  carefully  as  possible  with  a  moist  cloth 
or  a  piece  of  lens  paper.  Place  four  minute  drops  of  cement  care- 
fully at  the  edge  of  the  cover  at  the  four  quarters  and  allow  them  to 
harden  for  half  an  hour  or  more  ;  these  will  anchor  the  cover-glass 
and  the  preparation  may  then  be  placed  upon  the  turn-table  and  a 
ring  of  shellac  cement  put  round  the  edge  while  revolving  the  turn- 
table. 

§ni.  Sealing  glycerin-jelly  mounts.  Allow  the  glycerin- 
jelly  to  harden  for  12  hours  or  longer.  With  a  knife  scrape  away 
the  superfluous  jelly  and  then  carefully  wipe  around  the  cover-glass 
with  a  cloth  moistened  with  water.  Place  the  slide  on  a  turn-table, 
carefully  center  the  cover-glass,  and  with  a  brush  seal  the  edge  of 
the  cover  by  a  ring  of  shellac  while  revolving  the  turn-table.  A 
second  coating  may  be  given  subsequently  if  needed,  after  the  first 
coat  has  dried. 

§  112.  Sealing  balsam  mounts.  This  is  necessary  only 
with  special  preparations,  and  should  in  any  case  be  done  only  after 
the  preparations  have  dried  out  for  several  weeks.  With  a  knife 
scrape  off  all  superfluous  balsam  from  around  the  cover-glass  and 
wipe  it  carefully  with  a  cloth  moistened  with  alcohol  or  benzin  (or 
xylene).  Seal  as  with  glycerin-jelly  mounts.  When  the  oil  im- 
mersion is  to  be  used  often,  it  is  advantageous  to  seal  the  prepara- 
tion with  shellac  (after  it  has  dried)  to  facilitate  cleaning  away  the 
immersion  fluid. 

LABELING    MICROSCOPIC   SLIDES. 

§  113.  Every  permanent  microscopic  preparation  should  be 
carefully  and  neatly  labeled  in  ink,  the  label  being  placed  upon  the 
right  hand  end  of  the  slide.  The  label  should  furnish  at  least  the 
following  information  : 

EXAMPLE. 


(1)  The  number  of  the  preparation, 

the  thickness    of   the  cover- 
glass  and  of  the  section. 

(2)  The  name,  kind,  and  source  of  m 

Transection. 
the  preparation. 


No.  C.  15. 

S. 


Ileum  of  Cat. 


(3)  The  date  of  the  specimen. 


November,  1898. 


125 

In  the  case  of  specimens  with  which  it  is  advantageous  to  have 
more  information  at  hand  a  second  label  may  be  placed  upon  the 
other  end  of  the  slide,  and  it  may  bear  the  following  information  : 

(1)  Mode  of  fixation. 

(2)  Imbedding  method. 

(3)  Stains  employed. 

(4)  Mounting  medium  (generally  not  necessary). 

(5)  Special  purpose  of  the  preparation. 

A  catalog  giving  the  full  data  of  the  specimen, — age,  condition 
of  the  animal,  mode  of  preparation  in  detail,  special  points  illus- 
trated, etc.,  is  not  required  for  the  preparations  in  this  course,  but 
is  valuable  particularly  in  special  investigations  and  with  standard 
specimens. 

CLEANING   SLIDES   AND   COVER-GLASSES. 

§  114.  Cleaning  cover-glasses.  Fill  the  large  glass  box 
one-half  full  of  cleaning  mixture  and  put  in  the  new  covers, 
one  at  a  time,  being  sure  that  they  are  entirely  immersed  and  the 
cleaning  mixture  reaches  all  points.  The  one  cover  may  be  pushed 
under  by  the  next.  Let  them  remain  over  night  (or  longer)  and 
then  wash  them  thoroughly  in  running  water,  until  all  trace  of  the 
cleaning  mixture  is  removed.  Then  place  the  covers  in  50  or  6j% 
alcohol. 

§  115.  Wiping  the  cover-glasses.  When  ready  to  wipe  the 
cover-glasses,  remove  several  from  the  alcohol  and  put  them  on  a 
soft,  dry  cloth,  or  on  some  of  the  lens  paper  to  let  them  drain. 
Grasp  a  cover-glass  by  its  edges,  cover  the  thumb  and  index  finger 
of  the  other  hand  with  a  soft,  clean  cloth,  or  some  of  the  lens  paper. 
Grasp  the  cover  between  the  thumb  and  index  finger  and  rub  the 
surfaces.  In  doing  this  it  is  necessary  to  keep  the  thumb  and  index 
well  opposed  on  directly  opposite  faces  of  the  cover  so  that  no  strain 
will  come  on  it,  otherwise  the  cover  is  liable  to  be  broken. 

When  a  cover  is  well  wiped,  hold  it  up  and  look  through  it  to- 
ward some  dark  object.  The  cover  will  be  seen  partly  by  transmit- 
ted and  partly  by  reflected'  light,  and  any  cloudiness  will  be  easily 
detected.  If  the  cover  does  not  look  clear,  breathe  on  the  faces  and 
wipe  again.  If  it  is  not  possible  to  get  a  cover  clear  in  this  way  it 
should  be  put  again  into  the  cleaning  mixture.  When  the  covers 


126 

are  wiped,  put  them  in  a  clean  glass  box.  Handle  them  always  by 
their  edges,  or  use  fine  forceps.  Do  not  put  the  fingers  on  the  faces 
of  the  covers  for  that  will  surely  cloud  them. 

§  116.  Measuring  the  thickness  of  the  cover-glasses. 
With  the  cover-glass  measurer  determine  the  thickness  of  the  cover- 
glasses  and  sort  them  into  three  groups  :  (a)  those  with  a  thick- 
ness of  .  13-.  17  mm.,  (£)  those  less  than  .13  mm.,  and  (c)  those 
thicker  than  .17  mm.  Groups  (a)  and  (£)  only  should  be  used  ;  (V) 
should  be  discarded  or  used  only  with  objects  for  low  magnification. 

It  is  advantageous  to  know  the  thickness  of  the  cover-glass  on 
an  object  for  the  following  reasons  :  (a)  That  one  do  not  try  to  use 
objectives  in  studying  the  preparation  of  a  shorter  working  distance 
than  the  thickness  of  the  cover-glass  (Microscopical  Methods,  §  57)  ; 
(£)  In  using  adjustable  objectives  with  the  collar  graduated  for  dif- 
ferent thicknesses  of  cover,  the  collar  might  be  set  at  a  favorable 
point  without  loss  of  time  ;  (c)  For  unadjustable  objectives  the 
thickness  of  cover  may  be  selected  corresponding  to  that  for  which 
the  objective  was  corrected  (see  Microscopical  Methods  §  27  table). 
Furthermore  if  there  is  a  variation  from  the  standard  one  may  rem- 
edy it  in  part  at  least  by  lengthening  the  tube  if  the  cover  is  thinner 
and  shortening  it  if  the  cover  is  thicker  than  the  standard  (Micr. 
Meth.  §  96). 

§117.  Cleaning  slides.  Rinse 'new  slides  thoroughly  in 
clean  water  and  then  wipe  them  with  a  soft  towel.  In  cleaning  the 
slides  handle  them  by  their  edges  to  avoid  soiling  the  face  of  the 
slide.  After  the  slides  are  cleaned,  to  keep  them  free  from  dust 
store  them  again  in  the  box  in  which  they  came,  or  in  a  covered 
glass  dish  or  jar. 

§  118.  Cleaning  used  slides  and  covers.  If  only  water, 
glycerin  or  glycerin -jelly  has  been  used  on  them,  they  may  be  cleaned 
with  water,  preferably  warm  water,  and  then,  if  necessary,  wiped 
out  of  50%  alcohol.  If  balsam  has  been  used,  heat  the  slides  until 
the  balsam  is  soft  and  then  remove  the  cover-glasses.  Scrape  from 
the  slides  all  the  balsam  possible  and  place  them  (and  cove»glasses) 
in  cleaning  mixture  for  several  days.  If  then  they  cannot  be  readily 
cleaned,  place  them  in  fresh  cleaning  mixture  for  a  period  of  several 
days.  Wash  away  the  cleaning  mixture  thoroughly  with  water  and 
wipe  them  with  a  clean  towel. 


127 
SPECIAL  METHODS. 

THE   BLOOD. 

§  1 19.  Special  methods  in  the  examination  of  the  blood  include 
(i)  Examining  fresh  ;  (2)  Technic  of  staining  blood  films;  (3) 
Determination  of  the  number  of  red  and  white  corpuscles  per  cubic 
millimeter  ;  (4)  Determination  of  the  relative  amount  of  hemoglo- 
bin ;  (5)  Spectroscopic  examination  of  blood  (hemoglobin),  (i) 
and  (2)  are  briefly  given  here  ;  for  (5)  see  Microscopical  Methods, 
§§  201-203. 

§  120.  Examining  fresh.  This  consists  in  covering  a  drop 
on  a  slide  and  immediately  sealing  the  cover-glass  to  prevent  evapo- 
ration, observing  the  following  cautions:  (i)  The  drop  of  blood 
(from  the  finger  or  the  lobe  of  the  ear)  should  flow  freely  and  not 
be  obtained  by  pressure.  The  drop  should  be  a  medium-sized  one, 
which  will  spread  out  in  an  even,  thin  layer  under  the  cover.  (2) 
The  drop  should  be  received  upon  a  cover  or  slide,  covered,  and 
sealed  at  once  with  castor  oil. 

Examination  of  fresh  blood  may  be  used  in  clinical  examination 
for  the  detection  of  some  abnormal  conditions,  and  it  is  of  value  in 
the  rough  diagnosis  of  many  others. 

§  121.  Stained  preparation  of  blood,  (a)  Preparing  the 
blood  film.  This  may  be  best  done  in  one  of  two  ways  :  (i)  The 
edge  of  a  slide  is  first  drawn  through  a  drop  of  fresh  blood  and  then 
moved  quickly  across  the  surface  of  a  clean  cover-glass,  in  this  way 
spreading  the  blood  in  a  thin,  even  layer  upon  the  cover.  Success 
depends  upon  getting  the  right  amount  of  blood  upon  the  edge  of 
the  slide  and  the  quick,  even  movement  by  which  it  is  spread  upon 
the  cover-glass.  A  second,  possibly  better,  method  is  the  following  : 

(2)  Have  ready  two  thin  clean  cover-glasses  and  obtain  a  drop 
of  fresh  blood.  Take  one  of  the  covers  in  the  forceps,  touch  it  to 
the  drop  of  blood  and  place  it  upon  the  second  cover-glass  eccentric- 
ally, with* one  edge  projecting  slightly.  Slip  the  two  covers  apart 
in  the  plane  of  their  surfaces  and  dry  them  quickly  by  waving  them 
in  the  air  or  by  passing  them  rapidly  over  the  tip  of  a  flame.  The 
lower  cover-glass  will  have  the  better  film. 

(£)  Fixing  the  hemoglobin  with  (a)  ether-alcohol  or  (b)  heat. 


128 

§  122.  Fixing  with  ether-alcohol.  When  the  blood  films 
on  the  covers  are  dry,  place  them  in  ether-alcohol  (equal  parts)  for 
YZ-I  or  several  hours.  Let  them  fix  for  a  longer  rather  than  a 
shorter  time,  as  the  quality  of  the  stain  (with  triacid  mixture)  will 
be  improved.  After  they  have  fixed  a  sufficient  time  remove  and 
again  dry  them  in  the  air.  They  may  now  be  stained,  immediately 
or  at  convenience. 

§  123.  Fixing  with  heat.  Place  a  gas  or  alcohol  flame  under 
the  apex  of  a  triangular  copper  tabte  (or  other  similar  warm  table  or 
incubator).  When  it  is  well  heated  determine  the  region  that  has 
a  temperature  equal  to  the  boiling  point  of  water  (100°  C)  by  plac- 
ing on  it  drops  of  water  at  varying  distances  from  the  flame.  Just 
within  the  point  so  determined  (nearer  the  flame)  place  the  covers 
bearing  the  dried  film  of  blood,  film  side  down  upon  the  copper 
plate.  Leave  them  for  15-30  minutes  or  longer.  When  the  covers 
have  cooled  they  are  ready  to  be  stained. 

(<r)  Staining  with  (#)  Eosin  and  Hematoxylin,  (b}  Hhrlich's 
'Triacid  Mixture,  or  (c)  Methylene  Blue. 

§  124.  Eosin  and  hematoxylin.  Stain  the  fixed  blood  films 
for  2-3  minutes  with  a  l/2%  aqueous  solution  of  eosin,  rinse  with 
water  and  stain  for  10-15  minutes  with  hematoxylin,  rinse  again 
with  distilled  water  and  allow  the  film  to  dry.  When  dry,  warm  the 
cover  gently  to  remove  the  last  traces 'of  moisture  and  mount  in 
balsam.  This  gives  a  good  general  stain  of  the  blood  corpuscles, 
both  red  and  white.  Red  corpuscles  will  be  stained  red  or  pink, 
the  nuclei  of  the  white  corpuscles  will  be  blue,  and  their  cell  bodies 
but  faintly  stained  ;  eosinophile  granules,  a  bright  pink. 

§  125.  Ehrlich's  triacid  mixture  (§  93).  Place  a  drop  or 
two  of  the  mixture  upon  the  film  side  of  the  cover.  Stain  for  10-15 
minutes,  rinse  in  distilled  water,  dry,  and  mount  in  balsam. 

If  the  stain  is  successful,  red  corpuscles  will  be  orange-yellow, 
nuclei  of  white  corpuscles,  blue,  cell  bodies,  pale  pink,  eosinophile 
granules,  copper  red.  This  stain  is  used  a  great  deal  in  the  clinical 
examination  of  blood  and  is  a  valuable  one.  Should  the  red  corpus- 
cles be  colored  red  instead  of  orange-yellow,  it  means  that  the  fixa- 
tion has  fjeen  insufficient  ;  if  ether-alcohol  was  used  for  fixing,  the 
time  was  too  short  ;  if  heat  was  employed,  the  degree  of  heat  was 
insufficient  or  the  time  too  short.  The  right  time  of  fixation  and 
staining:  must  be,  in  many  cases,  determined  by  experiment. 


129 

§  126.  Methylene  blue.  Formula  :  Methylene  blue,  satu- 
rated alcoholic  solution,  i  part  ;  distilled  water,  2  parts. 

This  is  for  staining  "  basophile  "  granules  in  mast  cells.  Prep- 
arations fixed  by  heat  or  ether-alcohol  are  stained  15  minutes  or  so, 
washed  with  water,  dried,  and  mounted  in  balsam.  Nuclei  and  bas- 
ophiie  granules  stain  blue  ;  all  else  is  colorless,  or  nearly  so.  This 
stain  has  but  little  value  in  the  study  of  normal  blood,  since  baso- 
phile leucocytes  are  of  rare  occurrence. 

FINE   INJECTION. 

For  the  purpose  of  examining  microscopically  the  finer  arteries 
and  veins  and  the  capillaries  in  a  tissue,  and  their  relation  to  the 
other  parts,  it  is  necessary  to  fill  them  with  some  colored  injection 
mass,  or  otherwise  stain  or  color  them.  The  masses  employed  for 
the  fine  injection  of  tissue  in  this  course  are  Carmine  Gelatin  Mass 
(red)  and  Berlin  Blue  Gelatin  Mass. 

§  127.  Carmine  gelatin  mass.  Formula  :  Dry  gelatin,  75 
grams  ;  carmine  (No.  40),  10  grams  water,  90  c.c.  ;  ammonia,  10 
c.c.  ;  acetic  acid,  q.  s.  ;  chloral  hydrate,  10  grams. 

Soak  the  gelatin  in  water  until  it  is  soft  ;  pour  off  the  superflu- 
ous water  and  melt  it  (in  an  agate  or  porcelain  dish)  over  a  water 
bath.  Grind  the  carmine  to  a  paste  with  water  ;  add  all  the  am- 
monia and  water  ;  filter,  warm  to  80°  or  90°  C. ,  and  add  to  the  warm 
gelatin.  Then  add  slowly  the  acetic  acid  diluted  with  an  equal  vol- 
ume of  water,  while  constantly  stirring  the  mass,  until  the  mass 
smells  very  slightly  of  the  acid.  Filter  through  fine  flannel.  If  the 
mass  is  acid,  the  chloral  hydrate  may  be  safely  added  (as  a  preserva- 
tive) ;  if  any  ammonia  is  present  it  will  decompose  it  forming  chlo- 
roform and  a  granular  precipitate.  If  too  much  acid  is  added,  the 
gelatin  will  not  set. 

§  128.  Berlin  blue  injection  mass.  Formula:  Dry  gela- 
tin, 75  grams  ;  saturated  aqueous  solution  of  Berlin  blue,  150  c.c. 
chloral  hydrate,  10  grams.  Prepare  the  gelatin  in  the  manner  given 
above  (§  127)  ;  warm  the  Berlin  blue  solution  (to  80°  or  90°  C.), 
and  add  it  to  the  hot  gelatin.  Heat  the  mixture  for  10  minutes  or 
more,  stirring  it  occasionally,  and  filter  it  through  fine  flannel  and 
add  the  chloral  hydrate. 


130 

§  129.  For  securing  the  best  results  in  injecting  the  following 
conditions  should  be  observed  :  ( i )  A  young  but  nearly  mature, 
lean  animal  is  to  be  preferred.  (2)  Kill  the  animal  with  an  anes- 
thetic (chloroform;  and  leave  it  in  the  anesthetic  at  least  half  an 
hour  before  beginning  the  injection  ;  do  not,  however,  wait  until 
rigor  mortis  sets  in.  (3)  Inject  only  the  part  desired,  tying  all  an- 
astomosing vessels  and  all  vessels  to  other  parts.  Inject  into  the 
artery  of  the  part,  leaving  the  vein  open  until  nearly  pure  injection 
mass  escapes,  then  tie  it  and  continue  the  injection  until  the  part 
feels  hard  and  is  the  color  of  the  injection  mass.  (4)  When  the 
injection  is  finished  cool  the  part  injected  by  means  of  cold  water, 
ice,  or  snow. 

(5)  Harden  the  injected  tissue  i  or  2  days  in  50%  alcohol,  2 
or  3  days  in  67%  and  82%  alcohols.  The  acidity  of  the  alcohols 
should  be  insured  by  adding  to  the  50%  alcohol  a  few  drops  of  acetic 
acid.  The  tissue  may  be  .stored  in  82,%  alcohol  until  ready  for  sec- 
tioning. For  sectioning  the  collodion  method  is  usually  preferable. 

§  130.  Silvering  blood  vessels.  Silver  nitrate  may  be  used 
for  coloring  blood  vessels,  and  thus  differentiating  them.  See  §  147. 

CALCIFIED   STRUCTURES, — BONE   AND   TOOTH. 
(A).     Decalcification. 

§  131.  For  the  purpose  of  investigating  the  soft  structures  of 
tissues  containing  lime  salts,  such  as  bone,  teeth,  and  calcified  carti- 
lage, it  is  necessary  to  remove  the  lime  salts  before  sections  can  be 
prepared  in  the  usual  way  by  a  process  known  as  decalcified  tion. 
Solutions  of  a  large  number  of  acids,  combined  or  uncombined  with 
other  substances,  may  be  used  as  decalcifiers.  Very  satisfactory 
are:  (i)  nitric  acid,  3  c.c.  ;  70%  (67%)  alcohol,  97  c.c.,  and  (<?) 
nitric  acid,  5  c.c.  ;  saturated  aqueous  solution  of  (potash)  alum,  50 
c.c.  ;  water,  50  c.c.  In  the  first  formula  the  alcohol,  in  the  second 
the  alum  acts  as  a  restrainer  of  the  nitric  acid.  The  first  of  these 
formulas  is,  perhaps,  better  for  bone  ;  the  second  has  a  more  rapid 
action  and  is  possibly  a  better  decalcifier  for  teeth. 

§  132.  Directions  for  use.  The  tissue  to  be  decalcified  must 
be  first  thoroughly  fixed  and  hardened  by  one  of  the  approved 
methods, — picric  alcohol  is  quite  satisfactory, — and  should  be  in 


82%  alcohol.  In  fixing,  structures  not  needed  should  be  removed,— 
muscles  trimmed  away  from  the  bone,  etc.  Bones  or  teeth  should 
be  opened  with  nippers  or  a  saw,  so  that  the  fluid  may  reach  the 
marrow  or  pulp  cavity. 

Place  the  hardened  tissue  in  the  decalcifier,  where  it  should 
remain  until  the  lime  salts  have  been  entirely  removed,  as  may  be 
ascertained  by  inserting  a  fine  needle  ;  if  any  calcified  matter  remains 
there  will  be  a  gritty  feeling  on  using  the  needle.  The  time  neces- 
sary for  complete  decalcification  will  depend  upon  the  size  and  den- 
sity of  the  calcified  tissue,  and  will  vary  from  3  to  15  days  or  longer. 
The  decalcifier  should  be  changed  after  the  first  day,  and  if  the  tis- 
sue is  large  it  is  best  to  change  it  subsequently  two  or  three  times  at 
intervals  of  two  or  three  days. 

When  decalcification  is  complete  rinse  the  tissue  well  in  water 
for  a  few  minutes  and  place  it  in  67%  alcohol  for  one  or  two  days 
and  then  in  82%  alcohol  for  several  days,  or  until  ready  to  imbed. 
The  82%  alcohol  should  be  changed  once  or  twice  in  order  that  the 
nitric  acid  may  be  well  washed  out.  Although  paraffin  in  many 
cases  may  be  employed  for  imbedding,  the  collodion  method  is  gen- 
erally more  satisfactory. 

Hematoxylin  with  eosin,  hematoxylin  with  picrofuchsin,  and 
hematoxylin  with  picrocarmine  afford  good  stains ;  by  staining 
thoroughly  with  hematoxylin  a  differential  staining  of  bone  and 
cartilage  may  be  obtained. 

(/?).     Sections  of  Dry  Bone  or  Tooth. 

§  133.  Though  the  general  structure  of  bone  and  teeth  is 
shown  moderately  well  when  the  tissue  has  been  decalcified  (§  131), 
the  Haversian  canals,  canaliculi  and  lacunae  of  bone  and  the  denti- 
nal  tubules  of  the  teeth  are  shown  much  better  in  sections  of  dried, 
non-decalcified  tissue,  rendered  sufficiently  thin  for  microscopic  ex- 
amination by  grinding  or  filing. 

§  134.  Directions  for  procedure.  Prepare  thin  transverse 
sections  of  dried  bone  in  accordance  with  the  directions  below. 
Longitudinal  (radial)  sections  and  tangential  (surface)  sections  may 
also  be  prepared  in  the  same  manner,  the  former  to  show  the  Haver- 
sian canals  and  their  anasomoses,  the  latter  to  indicate  the  shape  of 
the  lacunae  as  seen  in  a  different  plane. 


132 

1.  Sawing  the  section.     Make  an  exact  transection  of  a  part  of 
the  shaft  of  a  long  bone.     The  section  should  be  about  i  cm.  long 
and  include  the  thickness  of  the  shaft  from  the  surface  to  the  medul- 
lary cavity.     Make  the  sections  about  i  mm.  thick. 

2.  Grinding  the  sections.     Place  the  piece  of  bone  on  a  cork  or 
piece  of  soft  wood  and  wet  it  with  water.     File  it  on  one  side  until 
smooth  and  then  turn  it  over.     Continue  the  filing  till  the  piece  is 
from  .05  to  .10  mm.  thick,  using  the  cover-glass  measurer  to  deter- 
mine the  thickness.     In  the  beginning  one  can  press  quite  hard  in 
filing  ;  as  the  section  thins,  more  care  should  be  exercised  and  the 
pressure  should  lessen. 

3.  Washing  and  drying  the  section.     When  the  section  is  thin 
enough,  rinse  it  and  dry  it  with  lens  paper. 

4.  Mounting  the  sections  in  hard  balsam.     To  prepare  the  bal- 
sam, put  two  or  three  large  drops  on  the  middle  of  a  slide  and  heat 
the  slide  in  some  way  to  drive  off  the  volatile  constituents.     Do  not 
heat  the  balsam  hot  enough  to  produce  bubbles.     When  the  balsam 
chips  after  cooling,  it  is  ready  for  use. 

In  mounting,  have  the  section  and  a  clean  cover  so  placed  that 
they  may  be  easily  and  quickly  grasped.  A  cork  somewhat  smaller 
than  the  cover-glass  should  be  within  reach,  and  also  a  stone  or 
piece  of  glass  upon  which  to  quickly  cool  the  specimen  as  soon  as  it 
is  mounted. 

Heat  the  slide  until  the  balsam  is  well  melted.  Put  the  slide 
upon  a  piece  of  paper,  grasp  the  piece  of  bone  with  the  forceps  and 
plunge  it  into  the  melted  balsam  ;  put  on  the  cover  as  quickly  as 
possible  and  press  it  down  with  the  cork  ;  finally  put  the  slide  on 
the  stone  or  glass  to  cool  the  balsam  quickly.  All  of  this  should  be 
done  as  rapidly  as  possible,  and  if  done  rapidly,  the  air  will  be 
retained  in  the  lacunae  and  canaliculi,  and  cause  them  to  stand  out 
as  black  spots  and  lines.  If  soft  balsam  were  used  it  would  soon 
drive  out  the  air,  and  being  of  nearly  the  refractive  index  of  bone,  it 
would  obliterate  the  lacunae  and  canaliculi.  Further,  if  the  hot  bal- 
sam were  not  cooled  quickly,  the  air  would  be  driven  out  and  balsam 
would  take  its  place  in  the  spaces. 

CENTRAL   NERVOUS   SYSTEM. 

§  135.  There  are  two  special  groups  of  methods  employed  in 
the  microscopical  examination  of  the  central  nervous  system,  (a) 


133 

Weigert  Hematoxylin  Methods,  for  differentiating  my  clinic  nerve 
tracts,  and  (£)  the  Chrome- silver  Impregnation  Methods,  to  bring  out 
the  form  of  the  cells  and  differentiate  the  amyelinic  nerve-fibers. 

These  have  their  greatest  value  when  used  in  conjunction  with 
more  general  staining  methods,  which  supplement  the  first  and  give 
a  basis  for  the  correct  interpretation  of  the  appearances  produced  by 
the  second  method.  In  addition,  there  are  other  stains  helpful  in 
bringing  out  the  finer  structure  of  the  cells. 

§  136.  The  Weigert  hematoxylin  methods.  These  meth- 
ods all  involve  (a)  mordanting  the  tissue  with  a  chromium  or  cop- 
per salt,  (£)  staining  (over-staining)  with  a  strong  hematoxylin 
stain,  and  (r)  decolorizing  (differentiating)  the  sections  in  a 
bleacher  until  the  myelinic  fibers  are  blue,  all  else  (except  blood) 
white  to  brown. 

§  137.  The  following  method  is  serviceable  :  Harden  the  tis- 
sue in  a  3%  aqueous  solution  of  potassium  dichromate  for  about  2 
weeks,  and  in  a  5%  aqueous  solution  for  an  equal  period  (§  31). 
Imbed  in  collodion  ;  paraffin  may  be  used  if  the  piece  of  tissue  is 
small.  The  sections  should  not  be  thicker  than  25^.  Wash  the 
sections  in  water  (£§  68-70)  and  (a)  Mordant  them  1-12  hours  in  a 
half-saturated  aqueous  solution  of  copper  acetate ;  rinse  them 
well  in  water  and  (£)  Stain  them  for  1-24  hours  (until  black)  in 
Weigert's  hematoxylin  (§  83)  ;  rinse  well  in  water  and  (r)  Differ- 
entiate in  the  Weigert's  bleacher  (Formula:  Water,  200  c.  c. ; 
borax,  2  grams  ;  potassium  ferricyanide,  2.5  grams).  Watch  the 
differentiation  carefully  and  when  the  cinerea  has  become  a  golden 
brown  and  the  myelinic  fibers  a  rich  blue,  stop  the  action  and  wash 
the  sections  in  water,  running  or  changed  several  times,  for  at  least 
YZ  hour.  Dehydrate,  clear,  and  mount  in  alkaline  balsam  (best) 
(§158).  If  the  specimens  are  to  be  left  for  some  time  before  mount- 
ing, place  them  in  95%  alcohol  with  sodium  carbonate  added  to 
render  it  alkaline. 

§  138.  Pal's  method  may  be  used  if  it  is  desired  to  stain  the 
nerve  cells  subsequently. 

Harden  the  tissue  thoroughly  in  solutions  of  potassium  dichro- 
mate (3%  solution,  2  weeks  ;  5,%,  2  weeks  or  so).  Imbed  in  collo- 
dion (paraffin,  if  the  sections  are  small).  Stain  the  sections  in  Wei- 
gert's hematoxylin  (§83)  until  a  blue  black  ;  rinse  in  tap- water. 


134 

Treat  for  a  short  time — 20-30  seconds — with  a  %%  aqueous 
solution  of  potassium  permanganate  and  decolorize  in  Pal's  bleacher 
until  the  cinerea  is  grey  (white),  the  myelinic  fibers  blue.  The 
action  will  be  very  rapid  and  must  be  carefully  watched  ;  a  few  .sec- 
onds will  suffice.  Wash  the  sections  thoroughly  (*4  hour  or  more) 
in  running  water.  If  it  is  desired,  counter-stain  with  a  red  stain,— 
eosin,  erythrosin  or  carmine. 

This  method  is  not  as  reliable  as  the  method  first  given  ;  great 
care  must  be  exercised  in  the  differentiation  with  the  bleacher. 

§  139.  Chrome-silver  impregnation  methods.  In  this 
group  of  methods  the  tissue  is  hardened  a  certain  length  of  time  in  a 
fluid  containing  a  chrome  salt,  especially  potassium  dichromate,  and 
then  placed  in  an  aqueous  solution  of  silver  nitrate  (%%  and  y^% 
solutions  generally  employed).  If  successful,  the  cells  and  their 
processes,  amyelinic  and,  to  a  certain  extent,  myelinic  nerve  fibers, 
are  outlined  by  a  black  precipitate  (black  by  transmitted  light  ; 
brown  by  reflected  light). 

§  140.  Success  depends  on  (a)  the  kind  of  animal  ;  different 
parts  and  tissues  react  more  satisfactorily  in  some  animals  or  classes 
of  animals  than  in  others,  (b)  The  age  of  the  animal  ;  young  or 
fetal  animals  give  better  results  than  do  adults,  (c)  The  time  of 
hardening ;  it  is  necessary  that  the  tissue  be  hardened  a  certain 
length  of  time,  constant  (relatively)  for  a  certain  kind  of  tissue  under 
the  conditions  above  (a  and  fr).  It  is  necessary  that  the  correct  de- 
gree of  hardening  be  carefully  regulated,  (a?)  Different  organs  and 
regions  of  the  central  nervous  system  vary  greatly  in  the  ease  with 
which  they  can  be  made  to  furnish  satisfactory  impregnations.  Al- 
most certain  impregnations  of  hippocamp  can  be  gained  ;  cerebral 
cortex  is  likewise  quite  easy  to  stain.  With  the  olfactory  bulb  the 
action  is  not  constant  though  fairly  complete.  The  optic  lobes  and 
retina  of  birds  and  large  reptiles  are  more  satisfactory  than  those  of 
mammals.  The  myel  (spinal  cord)  of  embryo  birds  (7-14  day  chick 
best)  is  generally  more  satisfactory  than  that  of  mammals  ;  in  any 
case,  fetal  or  new-born  animals  should  be  employed.  Difficult  are 
satisfactory  impregnations  of  sympathetic  ganglia,  organs  of  special 
sense  and  the  intrinsic  nerves  of  the  viscera. 

§141.  Golgi's  Rapid  Method.  This  is  the  most  generally 
serviceable  of  the  different  methods. 


135 

§  142.  Directions  for  use.  Tissue  of  a  (preferably)  young  ani- 
mal is  placed  in  a  mixture  of  4  parts  of  3%  potassium  dichromate 
and  i  part  of  i%  osmic  acid.  The  amount  of  the  fluid  should  be  at 
least  twenty  times  the  bulk  of  the  tissue  and  should  be  changed  as 
soon  as  it  grows  turbid  or  loses  the  strong  characteristic  odor  of  the 
osmic  acid. 

After  the  hardening  has  proceeded  to  the  right  degree  (§  143), 
rinse  the  tissue  in  water  for  about  5  minutes  and  place  for  15  min- 
utes in  a  l/^%  solution  of  silver  nitrate,  and  then  for  2  or  more  days 
in  a  y±%  solution  of  silver  nitrate,  preferably  keeping  it  in  the  dark. 

Without  washing,  imbed  rapidly  in  collodion  as  follows  : 

(a)  Dehydrate  2-3  hours  in  95%  alcohol,  changed  two  or  three 
times  ;  (£)  place  in  thin  collodion  for  20  minutes,  in  thick  collodion 
for  20-30  minutes  ;  (c)  imbed  in  thick  collodion,  on  a  cork  or  block 
of  wood  (best;  §  55,  a)  ;  (d)  harden  the  mass  in  chloroform  for 
20-30  minutes,  and  (<?)  place  the  block  in  clarifier  and  cut,  sections 
being  50-100  //  thick,  according  to  the  nature  of  the  tissue  and  the 
character  of  the  impregnation. 

(/)  Place  the  sections  in  95%  alcohol  for  a  few  minutes  ;  clear 
in  carbol-xylene  and  mount  in  balsam  by  placing  the  section  on  the 
slide,  absorbing  the  clearer  thoroughly  by  means  of  tissue  paper  and 
spreading  over  it  thick  xylene  balsam.  Do  not  cover.  L,ater,when 
the  balsam  has  hardened  somewhat,  it  may  be  melted  by  heat  and 
much  of  the  superfluous  balsam  drained  from  the  section  and  scraped 
away  with  a  knife. 

§  143.  Time  of  hardening.  The  following  periods  will  prob- 
ably be  found  approximately  correct.  In  general  :  The  best  results 
are  to  be  obtained  with  kittens  3-20  days  old,  puppies  2  weeks  old, 
rats  8-10  days,  rabbits  8  days,  (a)  For  cerebral  cortex  (and  hip- 
pocamp)  :  New-born  kitten,  1-2  days  ;  kitten  half  grown  (3-4 
months),  3-4  days  ;  new-born  rabbit,  24  hours  ;  rabbit  one  month 
old,  2-3  days,  etc. 

(0)  For  spinal  cord  :  Chick  of  5-6  days'  incubation,  24  hours  ; 
chick,  14-15  days'  incubation,  3  days  ;  new-born  kitten  or  puppy, 
2-3  days. 

(c)  Cerebellum  :  New-born  kitten,  1-2  days  ;  kitten  half  grown, 
4  days. 

§  144.  Methylene  blue.  Imbed  in  paraffin  tissue  that  has 
been  hardened  in  95%  alcohol  ;  cut  sections  rather  thick,  15-20  //, 


1 36 

or  even  thicker.  Fasten  the  sections  to  the  slide  or  carry  them 
through  in  watch-glasses.  Stain  the  sections  in  a  .5%  aqueous  solu- 
tion of  methylene  blue  for  5-10  minutes,  heating  it  until  it  steams  ; 
rinse  in  water  and  dehydrate,  clear  in  oil  of  origanum  or  cajuput, 
and  mount  in  balsam.  The  nerve  cells  and  nuclei  will  be  stained 
blue,  all  else  colorless.  In  the  cell  bodies  the  corpuscles  of  Nissl 
will  be  stained.  Should  the  stain  be  not  selective  enough,  differen- 
tiate for  a  few  seconds  before  dehydrating  with  a  mixture  of  aniline 
oil,  i  part,  95%  alcohol,  9  parts. 

§  145.  General  methods,  vom  Rath's  and  Zenker's  fluids 
are  recommended  for  the  fixation  of  nervous  tissue  ;  either  paraffin 
or  collodion  may  be  used  in  imbedding  ;  Delafield's  hematoxylin 
with  picrofuchsin  (strong  formula,  b)  as  counter-stain  is  recom- 
mended for  staining,  though  other  hematoxylins  may  be  used  as  well. 

SILVER    NITRATE   IMPREGNATIONS. 

§  146.  The  preparations  stained  by  means  of  nitrate  of  silver 
were  prepared  as  follows  :  The  fresh  tissue  was  washed  well  for  a 
minute  or  so  in  distilled  water  to  remove  from  the  surface  all  album- 
inous substance,  and  then  transferred  for  2-5  minutes  or  longer  to  a 
y?%  aqueous  solution  of  silver  nitrate  ;  it  was  again  rinsed  in  water 
and  in  it  exposed  to  direct  sunlight  until  a  light  brown.  When,  by 
examination  with  the  microscope,  the  stain  was  found  to  be  suf- 
ficient it  was  again  rinsed  in  water  and  placed  in  glycerin  or  alco- 
hol. Employed  in  this  manner  with  fresh  tissue,  silver  nitrate 
stains  the  cell  cement,  affording  thus  negative  images  of  the  cells. 

§  147.  Silvering  Vascular  Epithelium.  In  order  that  the 
vasular  epithelium  of  small  arteries,  veins,  and  capillaries  should  be 
well  demonstrated,  silver  nitrate  solutions  of  l/£  to  **/%%  strength 
must  be  injected  into  the  vessels. 

§  148.  Procedure.  Connect  a  canula  with  the  artery  supply- 
ing the  alimentary  canal  (superior  mesenteric)  or  the  brain  (carotid) 
and  inject  distilled  water  until  the  water  flows  out  of  the  returning 
vein  colorless.  Then  immediately  inject  the  silver  solution  until  it 
runs  from  the  vein.  After  a  minute  or  two  follow  with  distilled 
water.  Place  the  intestines  and  mesentery  in  water  and  expose 
them  to  the  light  until  they  become  slightly  browned.  Strips  of  the 
muscular  coat  of  the  intestines,  especially  of  the  rabbit,  will  show 


137 

capillaries  well.  Veins  and  arteries  side  by  side  may  be  found  in 
the  mesentery.  If  the  brain  vessels  are  injected  one  can  get  admir- 
able preparations  showing  nuclei  as  well  as  cell  outline  by  staining 
in  hematoxylin.  Mount  in  glycerin,  or,  if  desired,  dehydrate  and 
mount  in  balsam.  The  tissue  may  be  kept  in  50%  alcohol  or  in 
50%  glycerin  for  several  months  before  mounting  if  it  is  kept  in  the 
dark. 

For  large  vessels  and  endocardial  epithelium  open  the  vessels 
or  the  heart  and  silver  as  directed  above  for  mesentery.  It  may  be 
necessary  to  make  thin  free-hand  sections  so  that  the  preparation 
will  be  thin  enough  for  high  powers. 

. 

FORMULAS. 

In  addition  to  those  given  elsewhere;  see  also  The  Microscope,  7th  ed., 
|§  297-316. 

2  149.  Acid  alcohol.  95%  alcohol,  100  c.c.  ;  hydrochloric  acid,  ^  c.c. 
For  Hcl  carmine,  use  95%  alcohol,  100  c.c.  ;  hydrochloric  acid,  y2  c.c. 

2  150.     Alcohol,     (i)  67%.     Take  95%  alcohol,  2  parts  ;  water,  i  part. 

(2)  82%.     Take  95%  alcohol,  5  parts  ;  water,  r  part. 

3  151.     Clarifier.     (Castor-xylene).     Castor  oil,  i  part ;  xylene,  3  parts. 

'i  152.  Clearer.  ( Carbol-xy lene ) .  Melted  carbolic  acid  crystals,  i  part 
(by  volume)  ;  xylene,  3  parts. 

\  153.  Normal  salt  solution.  NaCl.  (common  salt),  .6  gram  ;  distilled 
water,  100  c.c. 

$  154.  Collodion.  ( i )  Thick  collodion,  8%  solution.  Ether-alcohol,  100 
c.c.  ;  soluble  cotton,  8  grams.  (2)  6%  solution.  Ether-alcohol,  100  c.c.  ;  sol- 
uble cotton,  6  grams.  (3)  Thin  collodion,  i/4%,  ether-alcohol,  100  c.c.  ;  solu- 
ble cotton,  \yz  grams. 

§  155.     Ether-alcohol.     Sulphuric  ether,  i  paft ;  95%  alcohol,  i  part. 

2  156.  Glycerin,  eosin  and  alum  carmine.  Glycerin,  85  c.c.  ;  alum  car- 
mine, 7  ><  c.c.  ;  l/z  %  aq.  sol.  eosin,  7^  c.c. 

$  157.  Lampblack  mixture.  Lampblack,  i  gram  ;  gum  arable,  i  gram  ; 
common  salt,  T%  gram  ;  water,  20  c.c. 

$  158.  Neutral  (alkaline}  balsam.  Canada  balsam  is  liable  to  be  slightly 
acid.  This  is  of  advantage  for  mounting  sections  stained  with  carmine  or  with 
acid  fuchsin  (as  when  picro-fuchsin  is  used),  and  for  injected  preparations 
where  carmine  or  Berlin  blue  is  used  as  the  coloring  matter.  For  hematoxylin 
and  other  stains  easily  affected  by  acid  media  it  is  often  advantageous  to  use 
neutral  or  slightly  alkaline  balsam  as  a  mounting  medium.  To  obtain  this 
slightly  alkaline  balsam,  add  some  pure  sodium  carbonate  to  the  thin  xylene 
balsam  and  shake  thoroughly  at  intervals  for  a  day  or  so.  Allow  the  balsam  to 
stand  until  the  soda  has  settled,  then  decant  and  thicken  by  evaporation  till  of 
the  desired  consistency.  (  The  Microscope,  7th  ed.,  p.  176,  g  300). 


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